IgG stimulated remyelination of peripheral nerves

ABSTRACT

The present invention is based on the discovery of polyclonal IgG&#39;s ability to promote Schwann cell maturation, differentiation, and myelin production. Methods for treating non-idiopathic, demyelinating peripheral neuropathies in mammals, where the neuropathy is not immune-mediated or infection-mediated, through the administration of polyclonal IgG are provided. Types of demyelinating peripheral neuropathies treatable with the present invention include peripheral nerve trauma and toxin-induced peripheral neuropathies. Alternatively, a composition of polyclonal IgGs can be applied directly to a peripheral nerve cell to induce maturation, differentiation into a myelinating state, and myelin expression or promote cell survival.

CROSS REFERENCES TO APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/605,117 filed Feb. 29, 2012, the disclosure of which ishereby incorporated herein by reference in its entirety for allpurposes.

BACKGROUND OF THE INVENTION

Peripheral neuropathy is a manifestation of disorders that inflictdamage to the peripheral nervous system (PNS), a network of ganglia andneurons that transmit signals between the central nervous system (CNS),i.e. brain and spinal cord, and every other part of the body. Neurons ofthe PNS rely on Schwann cells for, e.g. myelination, accelerated nerveconduction, nerve development and regeneration, trophic support,production of nerve extracellular matrix, and modulation ofneuromuscular synaptic activity. These Schwann cells provide electricinsulation by wrapping a protein and lipid-rich myelin sheath aroundaxons of motor and sensory neurons. Given myelin's critical role, it isnot surprising that demyelination of peripheral axons is a hallmark ofacute and chronic peripheral neuropathies such as Guillain-Barrésyndrome (GBS), chronic demyelinating polyneuropathy (CIDP) andmultifocal motor neuropathy (MMN) as well as other peripheral nervepathologies induced by toxins, drugs or systemic diseases, e.g.diabetes.

Peripheral neuropathies can distort signal transmission, causingsymptoms that vary with the origin of the neuropathy and type or numberof nerves affected. For example, symptoms may depend on whether thedisorder affects sensory nerve fibers, which transmit sensoryinformation from the affected area to the CNS, or motor nerve fibers,which transmit impulses and coordinate motor activity from the CNS to amuscle, or both. Peripheral neuropathies can be classified asmononeuropathies, involving damage to one nerve, or polyneuropathies,involving damage of multiple nerves; acute, where symptoms appearsuddenly, progress rapidly, and resolve slowly, or chronic, wheresymptoms begin subtly, and progress slowly. Over 100 different types ofperipheral neuropathy have been identified to date. Clinical diagnosesof peripheral neuropathy can be made based on the clinical history ofthe subject, a physical examination, the use of electromyography (EMG)and nerve conduction studies (NCS), autonomic testing, and nervebiopsies, etc.

Current treatments for peripheral neuropathies are directed at theunderlying condition, where possible, and often used in conjunction withsymptomatic treatments, such as anti-inflammatory agents, painmanagement, mechanical aids, and/or surgical intervention, etc. The bodyalso possesses its own regenerative capacity in response to injury ordamage of the PNS. After injury to the PNS, Wallerian degeneration ofdistal nerve stumps occur, followed by Schwann cell degradation ofmyelin, phagocytosis of extracellular myelin, and recruitment ofmacrophages for further myelin clearance. Schwann cells can furtheradapt to pathological situations by its ability to dedifferentiate,proliferate, promote axonal regeneration and redifferentiate, andproduce myelin. See Bhatheja et al. (2006) Int. J. Biochem. Cell Biol.38(12):1995-9. In the course of repair, Schwann cells stimulate, guideaxonal regeneration, and target reinnervation, forming a regenerationtube of the axon, known as Bunger's band, by proliferating rapidly andproviding the axon with a path to grow along. See Burstyn-Cohen et al.(1998) J. Neurosci 18(21): 8875-8885. While functional nerveregeneration in the PNS can generally be observed (in contrast to CNSwhich lacks a regenerative mechanism for myelin clearance and axonregeneration), it is often limited or chronically impaired. Novel repairpromoting approaches for the PNS are therefore needed.

Recent studies on the CNS have yielded evidence of IgM's direct effecton oligodendrocytes, the myelinating glial cells of the central nervoussystem. For instance, targeting of oligodendrocyte-reactive IgMKantibodies to oligodendrocytes was found to promote CNS remyelination(Asakura et al., 1998). Other studies showed that treatment of anon-immune, toxin-induced model of demyelinating disease with pooledhuman IgM molecules results in a significantly enhanced oligodendrocytedifferentiation in the CNS (Bieber et al., 2000; Bieber et al., 2002;Warrington et al., 2007). The discovery of Fc receptors for IgM onoligodendrocytes, their precursor cells, and myelin in the CNS, offersfurther clues of a possible ligand-receptor interaction (Nakahara etal., 2003).

Knowledge gained from these oligodendrocyte-IgM studies, thoughmeaningful for CNS repair, fails to harness the regenerative capacity ofthe PNS (which contains no oligodendrocytes). In more relevant studies,administration of human IVIG was found to reduce disease duration in anEAN (autoimmune neuritis) rat model, simulating the PNS-specific,demyelinating Guillain-Barré syndrome (GBS) (Lin et al., 2007). Theeffects were postulated as being attributable to IVIG's immunomodulatoryrole and possible anti-inflammatory and secondary bystander axonal lossreduction capability. In a separate study of the humoral immune system,B-cell knockout JHD mice exhibited significant delay in macrophageinflux, myelin clearance, and axon regeneration after PNS injury. Rapidmyelin debris clearance was restored through passive transfer ofantibodies from naïve WT mice or anti-PNS myelin antibody, therebyconfirming the role of endogenous antibodies in promoting macrophageentrance and phagocytic activity (Vargas et al., 2010). Clinical trialswith administration of intravenous immunoglobulins (IVIG) have shownpositive effects for GBS, chronic demyelinating polyneuropathy (CIDP)and multifocal motor neuropathy (MMN), with the assumption thattreatment in each of these autoimmune or immune-mediated neuropathieswas accomplished through IVIG's immunomodulatory role.

The effect of polyclonal IgG on Schwann cells, if any, was heretoforeunknown. A question, therefore, remained as to how the regenerativefunction of Schwann cells could be harnessed for therapeutic purposes indemyelinating, peripheral neuropathies. The present discovery ofexogenous polyclonal IgG's ability to induce Schwann cell maturation,differentiation, and myelin production, is an important clarification ofmechanism that provides novel approaches to the treatment of alldemyelinating peripheral neuropathies.

SUMMARY OF THE INVENTION

In one aspect of the invention, there is provided methods of treating ademyelinating peripheral neuropathy in mammals, wherein the neuropathyis not immune-mediated or infection-mediated, by administering atherapeutically effective amount of polyclonal IgG to a mammal diagnosedwith said neuropathy. In some embodiments of the invention, thedemyelinating peripheral neuropathy being treated is not Guillain-Barrésyndrome, chronic demyelinating polyneuropathy, or multifocal motorneuropathy. In other embodiments of the invention, the demyelinatingperipheral neuropathy is a non-idiopathic neuropathy. The demyelinatingperipheral neuropathy treatable by the present invention may be selectedfrom a trauma-induced neuropathy, a toxin-induced neuropathy, aninherited neuropathy, and a neuropathy induced by a metabolic disease,e.g. diabetic neuropathy.

In another aspect of the invention, there is provided methods oftreating peripheral nerve trauma by administering a therapeuticallyeffective amount of polyclonal IgG to a mammal with peripheral nervetrauma.

In yet another aspect of the invention, there is provided methods oftreating toxin-induced peripheral neuropathy, wherein the neuropathy isnot infection-mediated, by administering a therapeutically effectiveamount of polyclonal IgG to a mammal diagnosed with said neuropathy.

For treatment of a demyelinating peripheral neuropathy described herein,polyclonal IgG of the invention may be administered locally orsystemically. Local administration of the polyclonal IgG can occurintramuscularly or intradermally. Systemic administration of thepolyclonal IgG can occur intranasally, subcutaneously, orally,intra-arterially or intravenously. In some embodiments of the invention,an anti-inflammatory agent is co-administered with the polyclonal IgG tothe mammal. The anti-inflammatory agent may be selected from anadrenocorticotropic hormone, a corticosteroid, an interferon, glatirameracetate, or a non-steroidal anti-inflammatory drug.

The polyclonal IgG of the invention may be administered weekly,biweekly, or monthly at a dose of about 0.05 to 5 g per kg of patientbody weight or about 0.5 to 2 g per kg of patient body weight.

In a further aspect of the invention, there is provided methods ofpromoting myelination of a peripheral nerve cell by a Schwann cell bycontacting the Schwann cell with an amount of polyclonal IgG sufficientto promote myelination of said peripheral nerve cell by the Schwanncell.

In another aspect of the invention, there is provided methods ofpromoting the differentiation of an immature Schwann cell into amyelinating state by contacting said Schwann cell with polyclonal IgG inan amount sufficient to induce the Schwann cell differentiation.

In yet another aspect, there is provided methods of promoting myelinproduction by a Schwann cell comprising contacting said Schwann cellwith an amount of polyclonal IgG sufficient to upregulate MBP gene.

In a further aspect of the invention, there is provided methods ofculturing mammalian nervous tissue which comprises axons by contactingthe tissue in culture with an effective amount of Schwann cells and aneffective amount of polyclonal IgG, whereby the contacting of Schwanncells with polyclonal IgG induces upregulation of MBP gene.

In yet another aspect of the invention, there is provided methods oftreating a peripheral nerve injury in a mammal by: transplanting nervecells to a site of the peripheral nerve injury; and contacting the nervecells with a composition comprising Schwann cells and polyclonal IgG.

In the methods described herein, the polyclonal IgG can be given throughone or more routes of administration, such as intramuscularly,intradermally, subcutaneously, buccally, orally, intranasally, orintra-arterially or intravenously to an individual in need of suchtherapy. The individual may be a human or domesticated animal. In someembodiments, the polyclonal IgG is derived from pooled human serum.

In some embodiments, the polyclonal IgG Is co-administered with ananti-inflammatory agent to mammal in need of such therapy. Theanti-inflammatory agent may be selected from an adrenocorticotropichormone, a corticosteroid, an interferon, glatiramer acetate, or anon-steroidal anti-inflammatory drug.

In yet another aspect of the invention, there is provided pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier and aneffective amount of polyclonal IgG for treating a non-idiopathic,demyelinating peripheral neuropathy.

BRIEF DESCRIPTION OF THE DRAWINGS

More particular descriptions of the invention are made by reference tocertain exemplary embodiments thereof which are illustrated in theappended Figures. These Figures form a part of the specification. It isto be noted, however, that the appended Figures illustrate exemplaryembodiments of the invention and therefore are not to be consideredlimiting in their scope.

FIG. 1A-B shows the relative proliferation rates of immature Schwanncells that were exposed to nondialysed (FIG. 1A) and dialysed (FIG. 1B)IVIG/buffer formulations after 2 days as measured by BrdU incorporationassays. These relative proliferation rates were generated based on thenumber of cells positive for of 5-bromo-2′-deoxyuridine (BrdU)incorporated into cellular DNA during cell proliferation.

FIG. 2A-B shows the relative proliferative rates of immature Schwanncells that were exposed to nondialysed (FIG. 2A) and dialysed (FIG. 2B)IVIG/buffer formulations after 2 days as measured using Ki-67 assays.These relative proliferation rates were generated based on the number ofcells positive for Ki-67 expression during cell proliferation.

FIG. 3A-B shows the levels of P0 (FIG. 3A) and MBP (FIG. 3B) geneexpression in immature Schwann cells that were exposed to dialysedIVIG/buffer formulations at 1 day and 3 day time-points.

FIG. 4A-B shows the levels of P0 (FIG. 4A) and MBP (FIG. 4B) geneexpression in p57kip2 suppressed Schwann cells that were exposed todialysed IVIG/buffer formulations at the 7 day time-point (9 dayssuppression).

FIG. 5 shows the expression levels of CD64 Fc receptor in p57kip2suppressed Schwann cells as compared to control transfected Schwanncells (without p57kip2 suppression). Neither group of Schwann cells wereexposed to IVIG/buffer formulations.

FIG. 6A-B shows fluorescent images of p57kip2 suppressed Schwann cells(FIG. 6B) and control transfected cells (FIG. 6A) after stimulation with20 mg dialyzed IVIG/buffer formulations. The location and length ofcellular processes are indicated by the arrows superimposed onto thefluorescent images.

FIG. 7A-D shows a graph of the cell outgrowth length for p57kip2suppressed Schwann cells and control transfected cells (FIG. 7A) after 3days of stimulation with dialysed IVIG/buffer formulations (5 dayssuppression) along with the respective fluorescent images of the p57kip2suppressed Schwann cells stimulated with 20 mg of IVIG (FIG. 7B),p57kip2 suppressed Schwann cells stimulated with buffer (FIG. 7C),control transfected cells treated with 20 mg IVIG (FIG. 7D), and controltransfected cells treated with buffer (FIG. 7E).

FIG. 8A-D shows a graph of the cell outgrowth length for p57kip2suppressed Schwann cells and control transfected cells (FIG. 8A) after 7days of stimulation with dialysed IVIG/buffer formulations (9 dayssuppression) along with the respective fluorescent images of the p57kip2suppressed Schwann cells stimulated with 20 mg of IVIG (FIG. 8B),p57kip2 suppressed Schwann cells stimulated with buffer (FIG. 8C),control transfected cells treated with 20 mg IVIG (FIG. 8D), and controltransfected cells treated with buffer (FIG. 8E).

FIG. 9 is a flow diagram of the process for establishing a cocultue ofPNS neurons (rat dorsal root ganglion) and myelinating Schwann cells.

DETAILED DESCRIPTION OF THE INVENTION

The discovery of polyclonal IgG's ability to promote Schwann cellhomeostasis, maturation, differentiation, and myelin production can beapplied for treatment of demyelinating peripheral neuropathies ofvarying origins, e.g. toxin-induced neuropathies, diabetic neuropathy,trauma-induced neuropathy, by promoting the regenerative capacity ofnative Schwann cells. Contemplated is the administration of polyclonalIgG as an adjunct or replacement of existing therapeutic regimes orsymptomatic treatments for demyelinating peripheral neuropathies.Furthermore, the present invention can be used in the laboratory settingfor effecting peripheral nerve remyelination. Based on the findingsdescribed herein, polyclonal IgGs can be applied in nerve transplant,cell culture, e.g. induction of Schwann cell differentiation,determination of precursor cell fate, myelin gene regulation or proteinexpression, and as a pretreatment to or post-operative care regimen forsurgical techniques threatening or involving peripheral nerves.

I. DEFINITIONS

The term “non-idiopathic” refers to a disorder where the underlyingcause is known.

The term “peripheral neuropathy,” as used herein, refers to a disorderaffecting the peripheral nervous system, which excludes ganglion andnerves of the brain and the spinal cord. “Peripheral neuropathy” canmanifest as one or a combination of motor, sensory, sensorimotor, orautonomic neural dysfunction. The variety of morphologies exhibited byperipheral neuropathies can be attributed to a number of differentcauses. For example, peripheral neuropathies can be geneticallyacquired, can result from a systemic disease, or can be induced by atoxic agent. Examples include but are not limited to diabetic peripheralneuropathy, distal sensorimotor neuropathy, or autonomic neuropathiessuch as reduced motility of the gastrointestinal tract or atony of theurinary bladder. Examples of peripheral neuropathies associated withsystemic disease include post-polio syndrome or AIDS-associatedneuropathy; examples of hereditary peripheral neuropathies includeCharcot-Marie-Tooth disease, Abetalipoproteinemia, Tangier disease,Metachromatic leukodystrophy, Fabry's disease, and Dejerine-Sottassyndrome; and examples of peripheral neuropathies caused by a toxicagent include those caused by treatment with a chemotherapeutic agentsuch as vincristine, cisplatin, methotrexate, or3′-azido-3′-deoxythymidine.

One variety of peripheral neuropathy is “demyelinating peripheralneuropathy.” As used herein, a “demyelinating peripheral neuropathy”describes a broad class of peripheral neuropathies that are associatedwith the destruction or removal of myelin, the lipid-rich sheathsurrounding and insulating nerve fibers, from nerves. Non-limitingexamples of demyelinating peripheral neuropathy diseases includediabetic peripheral neuropathy, distal sensorimotor neuropathy, orautonomic neuropathies such as reduced motility of the gastrointestinaltract or atony of the urinary bladder. Further examples and descriptionsof demyelinating peripheral neuropathy can be found in Section II of theDetailed Description.

An “immune-mediated” disorder, as used herein, refers to a conditionwhich results from abnormal activity of the body's immune system.Subsets of “immune-mediated” disorder include, without limitation,autoimmune disease, wherein the immune system attacks the body,immune-complex disorders, disorders involving post-transplant rejection,inflammatory disease, and allergies.

An “infection-mediated” peripheral neuropathy refers to a dysfunction ofthe peripheral nervous system sustained as a result of viral, bacterial,or fungal infections.

A “trauma-induced peripheral neuropathy” or “traumatic peripheralneuropathy” refers to dysfunction of the peripheral nervous systemcaused by bodily shock, injury, or “physical trauma.” Physical trauma,e.g. from combat, vehicular accidents, falls, and sports-relatedactivities, can cause nerves to be partially or completely severed,crushed, compressed, or stretched, sometimes so forcefully that they arepartially or completely detached from ganglia or the spinal cord andresult in demyelination. Traum-induced peripheral neuropathies can alsobe sustained as a result of, e.g. electric shock, hypothermia, etc.

A “toxin” or “chemical induced” peripheral neuropathy refers todysfunction of the peripheral nervous system caused by toxins (e.g.,chemical agents). Toxins that produce peripheral neuropathy cangenerally be divided into three groups: drugs and medications;industrial chemicals; and environmental toxins. Non-limiting examples oftoxins that can cause peripheral neuropathy are described below inSection II of the Detailed Description.

An “anti-inflammatory agent” as used herein includes any agent thatreduces inflammation of an affected blood vessel and/or adjacent tissue.Non-limiting examples of anti-inflammatory agents are steroids (e.g.,glucocorticoids and corticosteroids), immune selective anti-inflammatoryderivatives (ImSAIDs), cooling agents, herbal supplements (e.g., devil'sclaw, hyssop, ginger, turmeric, arnica Montana, and willow bark(containing alicylilc acid), and foods with anti-inflammatory effects(e.g., pomegranate, green tea, vegetables, foods that contain omega-3fatty acids), nuts, seeds, and extra-virgin olive oil). Specifically,prostaglandin 2 (PGE2) is a pro-inflammatory compound and PGE1 and PGE3are anti-inflammatory compounds. Accordingly, agents that decrease PGE2or increase PGE1 and PGE3 can also act as anti-inflammatory agents.Additional non-limiting examples of anti-inflammatory agents can befound in Section VI, “Combination Therapy,” below.

An “immature Schwann cell,” as used herein, refers to a specific stagein the Schwann cell lineage. The first step along the Schwann celllineage gives the Schwann cell precursor, a proliferative cell thatbecomes associated with many axons and expresses the nerve growth factorreceptor (NGF-R), growth-associated protein 43 (GAP-32), and the neuralcell adhesion molecules N-CAM and L1. The subsequent “committed” Schwanncell is known as an immature Schwann cell; it becomes associated withprogressively fewer axons and expresses, in addition to the previouslynoted markers, S-100 protein (from this stage onward, all Schwann cellsexpress S-100). Committed Schwann cells develop into eithernonmyelinating Schwann cells, which remain associated with several axonsand express galactocerebroside (GalC) in addition to the previousmarkers, or into myelinating Schwann cells. Myelinating Schwann cellsprogress through a proliferative “premyelinating” stage, characterizedby transient expression of suppressed cAMP-inducible Pou-domaintranscription factor (SCIP), followed by a “promyelinating”GalC-positive stage, becoming associated with a single axon in theprogress. The final differentiation into a mature myelinating Schwanncell involves downregulation of NGF-R, GAP-43, N-CAM, and L1 expression,with upregulation of expression of GalC and myelin proteins, and invivo, the synthesis and elaboration of myelin.

The term “IgG,” as used herein, refers to a composition of IgGimmunoglobulins. The IgG class of immunoglobulins, as the name suggests,is characterized by the presence of a γ (gamma) heavy chain. Anexemplary whole IgG immunoglobulin structure comprises a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The N-terminus of each chain defines a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition. The terms variable light chain (V_(L)) and variable heavychain (V_(H)) refer to these light and heavy chains respectively.

An “immunoglobulin” or “antibody” is a polypeptide that isimmunologically reactive with a particular antigen. The term“immunoglobulin,” as used herein, encompasses intact molecules ofvarious isotypes as well as fragments with antigen-binding capability,e.g., Fab′, F(ab′)₂, Fab, Fv and rIgG. See, e.g., Pierce Catalog andHandbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J.,Immunology, 3.sup.rd Ed., W.H. Freeman & Co., New York (1998). The termalso encompasses recombinant single chain Fv fragments (scFv). The termfurther encompasses bivalent or bispecific molecules, diabodies,triabodies, and tetrabodies. Bivalent and bispecific molecules aredescribed in, e.g., Kostelny et al. (1992) J. Immunol. 148:1547, Packand Pluckthun (1992) Biochemistry 31:1579, Hollinger et al., 1993,supra, Gruber et al. (1994) J. Immunol.: 5368, Zhu et al. (1997) ProteinSci 6:781, Hu et al. (1996) Cancer Res. 56:3055, Adams et al. (1993)Cancer Res. 53:4026, and McCartney, et al. (1995) Protein Eng. 8:301.

The term “polyclonal IgG,” as used herein, refers to a heterogeneouscollection of IgG immunoglobulins derived from multiple B-cells andhaving different specificities and epitope affinities. Methods ofpreparing polyclonal antibodies are known to the skilled artisan (e.g.,Harlow & Lane, 1988, Antibodies: A Laboratory Manual. (Cold SpringHarbor Press)). The polyclonal IgGs of the invention can be extractedfrom plasma pooled from different mammalian individuals who have beenprescreened for pathogenic disorders. In some embodiments, thepolyclonal IgGs of the present invention are representative of over 100individuals, over 200 individuals, over 300 individuals, over 400individuals, over 500 individuals, over 600 individuals, over 700individuals, over 800 individuals, over 900 individuals, over 1000individuals, over 1100 individuals, over 1200 individuals, over 1300individuals, over 1400 individuals, over 1500 individuals, over 1600individuals, over 1700 individuals, over 1800 individuals, over 1900individuals, or over 2000 individuals.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein, in a heterogeneous population ofproteins and other biologics. Thus, under designated immunoassayconditions, the specified antibodies bind to a particular proteinsequences at least two times the background and more typically more than10 to 100 times background. A ligand (e.g., an antibody) thatspecifically binds to a protein generally has an association constant ofat least 10³ M⁻¹ or 10⁴ M⁻¹, sometimes 10⁵ M⁻¹ or 10⁶ M⁻¹, in otherinstances 10⁶ M⁻¹ or 10⁷ M⁻¹, preferably 10⁸ M⁻¹ to 10⁹ M⁻¹, and morepreferably, about 10¹⁰ M⁻¹ to 10¹¹ M⁻¹ or higher. A variety ofimmunoassay formats can be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select monoclonal antibodiesspecifically immunoreactive with a protein. See, e.g., Harlow and Lane(1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications,New York, for a description of immunoassay formats and conditions thatcan be used to determine specific immunoreactivity.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Myelin basic protein” (MBP), as used herein, refers to the gene as wellas the protein encoded thereby, which is a major protein component ofmyelin, comprising approximately 30% of the total protein content of themyelin sheath. MBP has been shown to be a major target autoantigen inMS, and T cells reactive with MBP play a key role in its pathogenesis(see, for example, Schwartz, R S, “Autoimmunity and Autoimmune Diseases”in Paul, Fundamental Immunology, 3rd Ed. Raven Press, New York, 1993,pp. 1033 1097; Brown and McFarlin 1981. Lab Invest 45, pp. 278 284;Lehmann et al. 1992. Nature 358, pp. 155 157; Martin et al. 1992. AnnRev Immunol 10, pp. 153 187; Sprent 1994. Cell 76, pp. 315 322; Su andSriram. 1991. J of Neuroimmunol 34, pp. 181 190; and Weimbs and Stoffel.1992. Biochemistry 31, pp. 12289 12296).

The term “axon” refers to an elongated fiber of a nerve cell responsiblefor conducting signals in the body.

The terms “individual,” “subject,” and “patient,” used interchangeablyherein, refer to a mammal, including, but not limited to, murines,simians, humans, mammalian farm animals, mammalian sport animals, andmammalian pets. In preferred embodiments, the individual is a human.

The terms “dose” and “dosage” are used interchangeably herein. A doserefers to the amount of active ingredient given to an individual at eachadministration. The dose will vary depending on a number of factors,including frequency of administration; size and tolerance of theindividual; severity of the condition; risk of side effects; and theroute of administration. One of skill in the art will recognize that thedose can be modified depending on the above factors or based ontherapeutic progress. The term “dosage form” refers to the particularformat of the pharmaceutical, and depends on the route ofadministration. For example, a dosage form can be in a liquid, e.g., asaline solution for injection.

A “therapeutically effective” amount or dose or “sufficient/effective”amount or dose, is a dose that produces effects for which it isadministered. The exact dose will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,Gennaro, Ed., Lippincott, Williams & Wilkins).

The term “treatment” or “therapy” generally means obtaining a desiredphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or condition or symptomthereof and/or may be therapeutic in terms of a partial or complete curefor an injury, disease or condition and/or amelioration of an adverseeffect attributable to the injury, disease or condition and includesarresting the development or causing regression of a disease orcondition. Treatment can also include prophylactic use to mitigate theeffects of injury, should it occur. For example, in one aspect, thepresent invention includes pre-administration to mitigate damage priorto surgery involving the peripheral nervous system. Treatment can alsorefer to any delay in onset, amelioration of symptoms, improvement inpatient survival, increase in survival time or rate, etc. The effect oftreatment can be compared to an individual or pool of individuals notreceiving the treatment.

A “control” is used herein, refers to a reference, usually a knownreference, for comparison to an experimental group. One of skill in theart will understand which controls are valuable in a given situation andbe able to analyze data based on comparisons to control values. Controlsare also valuable for determining the significance of data. For example,if values for a given parameter vary widely in controls, variation intest samples will not be considered as significant.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

II. DEMYELINATING PERIPHERAL NEUROPATHIES

The present invention is based on the discovery that polyclonal IgG canharness Schwann cells' regenerative capacity through stimulation ofSchwann cell maturation, differentiation, and myelin production. In thismanner, the invention targets a unifying mechanism of demyelinatingperipheral neuropathies so as to provide a broad-spectrum treatment forsuch disorders. For example, this invention targets demyelinatingperipheral neuropathies caused by physical trauma, toxic agents, anddiabetes.

Demyelinating disorders treatable by the polyclonal IgG compositiondescribed herein include, for example, peripheral neuropathies that aregenetically acquired, result from a systemic disease, or induced by atoxin or by trauma.

Genetic demyelinating neuropathies (also known as hereditaryneuropathies) are one of the most common inherited neurologicaldiseases. Genetic demyelinating neuropathies are divided into four majorsubcategories: 1) motor and sensory neuropathy, 2) sensory neuropathy,3) motor neuropathy, and 4) sensory and autonomic neuropathy.Specifically, the demyelinating hereditary neuropathies are oftenprogressive neuropathies with markedly decreased nerve conduction andvelocity and chronic segmental demyelination of the peripheral nerve.Gabreels-Festen et al., “Hereditary demyelinating motor and sensoryneuropathy,” Brain Pathol. 3(2):135-146 (1993). Examples of generalclasses of genetic deyelinating neuropathies include but are not limitedto diabetic peripheral neuropathy, distal sensorimotor neuropathy, orautonomic neuropathies such as reduced motility of the gastrointestinaltract or atony of the urinary bladder. Examples of hereditary peripheralneuropathies include Charcot-Marie-Tooth disease, Abetalipoproteinemia,Tangier disease, Metachromatic leukodystrophy, Fabry's disease, andDejerine-Sottas syndrome.

Systemic demyelinating peripheral neuropathies arise as side effects ofa systemic illness. Non-limiting examples of peripheral neuropathiesassociated with systemic disease include post-polio syndrome andAIDS-associated neuropathy. Furthermore, the following non-limitingsystemic diseases can have peripheral neuropathy symptoms: cancer,malnutrition, alcoholism, diabetes, AIDS, Lyme disease, Rheumatoidarthritis, chronic kidney failure, autoimmune disorders, hypothyroidism,and viral infections (e.g., hepatitis).

Toxin induced demyelinating peripheral neuropathies are caused byexposure to neurotoxic agents such as pharmaceutical agents, biologicalagents, and chemical exposure. Examples of toxins that cause peripheralneuropathies include, but are not limited to, chemotherapeutic agents(e.g., vincristine, paclitaxel, cisplatin, methotrexate, or3′-azido-3′-deoxythymidine), lead, mercury, thallium, organic solvents,pesticides, carbon disulfide, arsenic, acrylamide, diphtheria toxin,alcohol, anti-HIV medications (e.g., didanosine and zalcitabine),anti-tuberculosis medications (e.g., isoniazid and ethamubtol),antimicrobial drugs (e.g., dapsone, metronidazole, chloroquine, andchloamphenicol), psychiatric medications (e.g., lithium), radiation, andmedications such as amiodarone, aurothioglucose, phenyloin, thalidomide,coichicine, cimetidine, disulfiram, hydralazine, and high levels ofvitamin B6. Additional toxic agents that may cause peripheral neuropathyare listed below.

Trauma induced demyelinating peripheral neuropathies, as describedabove, are caused by bodily shock, injury, or physical trauma.

Accordingly, causes of peripheral neuropathy range widely, e.g. fromdiabetic complications; trauma; toxins including, without limitation,drugs and medications, industrial chemicals, and environmental toxins;autoimmune response; nutritional deficiencies; to vascular and metabolicdisorders. For example, demyelinating peripheral neuropathies may occuras a result of osteosclerotic myeloma, monoclonal protein-associatedperipheral neuropathy, hereditary motor and sensory peripheralneuropathies types 1 and 3, and hereditary susceptibility to pressurepalsies.

Similarly, symptoms of a demyelinating peripheral neuropathy also vary,e.g. with the type of nerves affected. For example, a human patienthaving a demyelinating disorder can have one or more symptoms of ademyelinating disorder such as, but not limited to, impaired vision,numbness, weakness in extremities, tremors or spasticity, heatintolerance, speech impairment, incontinence, dizziness, or impairedproprioception (e.g., balance, coordination, sense of limb position). Ahuman (e.g., a human patient) with a family history of a demyelinatingdisorder (e.g., a genetic predisposition for a demyelinating disorder),or who exhibits mild or infrequent symptoms of a demyelinating disorderdescribed above can be, for the purposes of the method, considered atrisk of developing a demyelinating disorder.

Specifically, sensory nerve damage caused by a demyelinating peripheralneuropathy can cause a more complex range of symptoms because sensorynerves have a wider, more highly specialized range of functions. Largersensory fibers enclosed in myelin (lipid-rich membrane folds that arespirally wrapped and insulate many nerves) register vibration, lighttouch, and position sense. Damage to large sensory fibers lessens theability to feel vibrations and touch, resulting in a general sense ofnumbness, especially in the hands and feet. Many patients cannotrecognize by touch alone the shapes of small objects or distinguishbetween different shapes. This damage to sensory fibers may contributeto the loss of reflexes (as can motor nerve damage). Loss of positionsense often makes individuals unable to coordinate complex movementslike walking or fastening buttons, or to maintain their balance whentheir eyes are shut. Neuropathic pain is difficult to control and canseriously affect emotional well-being and overall quality of life.

Smaller sensory fibers without myelin sheaths transmit pain andtemperature sensations. Damage to these fibers can interfere with theability to feel pain or changes in temperature. Individuals may fail tosense that they have been injured from a cut or that a wound is becominginfected. Others may not detect pains that warn of impending heartattack or other acute conditions. (Loss of pain sensation is aparticularly serious problem for individuals with diabetes, contributingto the high rate of lower limb amputations among this population.) Painreceptors in the skin can also become oversensitized, so that severepain is felt (allodynia) from stimuli that are normally painless.

Symptoms of autonomic nerve damage are diverse and depend upon whichorgans or glands are affected. Autonomic nerve dysfunction can becomelife threatening and may require emergency medical care in cases whenbreathing becomes impaired or when the heart begins beating irregularly.Common symptoms of autonomic nerve damage include an inability to sweatnormally, which may lead to heat intolerance; a loss of bladder control,which may cause infection or incontinence; and an inability to controlmuscles that expand or contract blood vessels to maintain safe bloodpressure levels. A loss of control over blood pressure can causedizziness, lightheadedness, or even fainting when an individual movessuddenly from a seated to a standing position (a condition known aspostural or orthostatic hypotension).

Gastrointestinal symptoms frequently accompany autonomic neuropathy.Nerves controlling intestinal muscle contractions often malfunction,leading to diarrhea, constipation, or incontinence. Individuals may alsoexperience difficulty eating or swallowing if certain autonomic nervesare affected.

The polyclonal IgG composition of the invention may also be used totreat demyelinating peripheral neuropathy which developed as acomplication of diabetes, i.e. Type I, Type II. Peripheral neuropathy isone of the major complications of diabetes. Both a decrease in nerveconduction velocity and increased resistance to conduction failurecaused by ischemia are among the earliest changes detected in diabeticpatients and animal models of the disease. Ultrastructural studies havedemonstrated changes in both axons and Schwann Cells (SC) (e.g.,decrease in axon caliber and segmental demyelination) as well as in themicrovasculature, all of which appear to develop independently. Somestudies concluded that the progressive loss of fibers in peripheralnerves observed in human diabetic neuropathy may be due, at least inpart, to delayed nerve degeneration and impaired nerve regeneration.Metabolic and microvascular abnormalities, as well as a deficiency inneurotrophins, have been considered responsible for the pathogenesis ofdiabetic neuropathy. The vascular alterations in diabetes consistsmainly of ischemia and endoneurial hypoxia. The mechanisms underlyingthese vascular abnormalities include degenerative changes in thesympathetic nerve endings of vasa nervorum, with the consequentimpairment in neural control of nerve blood flow and reduced productionof prostacyclin and nitric oxide in nerves.

Two distinct clinical manifestations of diabetic neuropathy are thoserepresented by patients suffering from painful symmetricalpolyneuropathy, and by patients with insensitive, painless feet. Thepainless neuropathy is the prevalent disorder and, according to severalstudies, is likely to reflect the degree of nerve degeneration. Thepainful syndrome, on the other hand, is associated with fewermorphological abnormalities. While it has also been proposed that thepainful syndrome may reflect nerve regeneration, as opposed todegeneration, several reports suggest that nerve regeneration isimpaired in diabetes. Analysis of several functional indices inperipheral sensory nerves of diabetic rodents also suggests depressed,rather than increased, function. For instance, experimental diabetesinduces several nociceptive responses including early thermalhyperalgesia that with time turns into hypoalgesia, mechanicalhyperalgesia, thermal and tactile allodynia, increased C fiber activityand reduced sensitivity to opioids. In this context, mechanicalhyperalgesia may result from increased firing after sustainedsuprathreshold mechanical stimulation of C fibers.

While therapies with antioxidants, vasodilators and neurotrophins mayreverse some functional and metabolic abnormalities in diabetic nerves,they only result in a partial amelioration of abnormal pain perception,suggesting that other pathways are at play. The present invention isable to promote Schwann cell's healing capacity towards treatment ofdiabetic neuropathy.

The polyclonal IgG composition of the invention may also be used totreat demyelinating peripheral neuropathy resulting from trauma. A“trauma-induced” neuropathy refers to damage to the nervous system fromexternal physical injury. Injury or sudden trauma, e.g. from warfare,automobile accidents, falls, and sports-related activities, can causenerves to be partially or completely severed, crushed, compressed, orstretched, sometimes so forcefully that they are partially or completelydetached from the spinal cord and result in demyelination. Less dramatictraumas also can cause serious nerve damage.

The polyclonal IgG composition of the invention may also be used totreat peripheral neuropathy caused by a toxic agent. Toxins that produceperipheral neuropathy can generally be divided into three groups: drugsand medications; industrial chemicals; and environmental toxins. As usedherein, the term “toxic agent” is defined as any substance that, throughits chemical action, impairs the normal function of one or morecomponents of the peripheral nervous system. The definition includesagents that are airborne, ingested as a contaminant of food or drugs, ortaken deliberately as part of a therapeutic regime.

The list of toxic agents that may cause peripheral neuropathy includes,but is not limited to, 3′-azido-3′-deoxythymidine, acetazolamide,acrylamide, adriamycin, alcohol, allyl chloride, almitrine,amitriptyline, amiodarone, amphotericin, arsenic, aurothioglucose,carbamates, carbon disulfide, carbon monoxide, carboplatin,chloramphenicol, chloroquine, cholestyramine, cimetidine, cisplatin,cis-platinum, clioquinol, colestipol, colchicine, colistin, cycloserine,cytarabine, dapsone, dichlorophenoxyacetic acid, didanosine;dideoxycytidine, dideoxyinosine, dideoxythymidine,dimethylaminopropionitrile, disulfuram, docetaxel, doxorubicin,ethambutol, ethionamide, ethylene oxide, FK506 (tacrolimus),glutethimide, gold, hexacarbons, hexane, hormonal contraceptives,hexamethylolmelamine, hydralazine, hydroxychloroquine, imipramine,indomethacin, inorganic lead, inorganic mercury, isoniazid, lithium,methylmercury, metformin, methotrexate, methylbromide, methylhydrazine,metronidazole, misonidazole, methyl N-butyl ketone, nitrofurantoin,nitrogen mustard, nitrous oxide, organophosphates, ospolot, paclitaxel,penicillin, perhexyline, perhexyline maleate, phenyloin, platinum,polychlorinated biphenyls, primidone, procainamide, procarbazine,pyridoxine, simvastatin, sodium cyanate, streptomycin, sulphonamides,suramin, tamoxifen, thalidomide, thallium, toluene, triamterene,trimethyltin, triorthocresyl phosphate, L-tryptophan, vacor, vincaalkaloids, vincristine, vindesine, megadoses of vitamin A, megadoses ofvitamin D, zalcitamine, zimeldine; industrial agents, especiallysolvents; heavy metals; and sniffing glue or other toxic compounds.

The polyclonal IgG composition of the invention may also be used totreat demyelinating peripheral neuropathy resulting from theadministration of chemotoxins for cancer therapy. Among the chemotoxinsknown to cause peripheral neuropathy are vincristine, vinblastine,cisplatin, paclitaxel, procarbazine, dideoxyinosine, cytarabine, alphainterferon, and 5-fluorouracil (see Macdonald, Neurologic Clinics 9:955-967 (1991)).

III. DIAGNOSIS AND MONITORING OF DEMYELINATING PERIPHERAL NEUROPATHIES

Diagnosis of demyelinating peripheral neuropathy can be made by aphysician or clinician using one or more methods known in the art. Aneurological examination is typically required and involves taking apatient history (including the patient's symptoms, work environment,social habits, exposure to any toxins, history of alcoholism, risk ofHIV or other infectious disease, and family history of neurologicaldisease), performing tests that may identify the cause of theneuropathic disorder, and conducting tests to determine the extent,site, and type of nerve damage.

A general physical examination and related tests may reveal the presenceof a systemic disease causing nerve damage. Blood tests can detectdiabetes, vitamin deficiencies, liver or kidney dysfunction, othermetabolic disorders, and signs of abnormal immune system activity. Anexamination of cerebrospinal fluid that surrounds the brain and spinalcord can reveal abnormal antibodies associated with neuropathy. Morespecialized tests may reveal other blood or cardiovascular diseases,connective tissue disorders, or malignancies. Tests of muscle strength,as well as evidence of cramps or fasciculations, indicate motor fiberinvolvement. Evaluation of a patient's ability to register vibration,light touch, body position, temperature, and pain reveals sensory nervedamage and may indicate whether small or large sensory nerve fibers areaffected.

Based on the results of the neurological exam, physical exam, patienthistory, and any previous screening or testing, additional testing maybe ordered to help determine the nature and extent of the neuropathy.Exemplary technologies for aiding in the diagnosis of peripheralneuropathies include: computed tomography scan, magnetic resonanceimaging, electromyography, nerve conduction velocity, nerve biopsy, orskin biopsy. Apparatuses useful in the diagnosis of peripheralneuropathies include, without limitation, U.S. Pat. No. 7,854,703.

Computed tomography, or CT scan, is a noninvasive, painless process usedto produce rapid, clear two-dimensional images of organs, bones, andtissues. X-rays are passed through the body at various angles and aredetected by a computerized scanner. The data is processed and displayedas cross-sectional images, or “slices,” of the internal structure of thebody or organ. Neurological CT scans can detect bone and vascularirregularities, certain brain tumors and cysts, herniated disks,encephalitis, spinal stenosis (narrowing of the spinal canal), and otherdisorders.

Magnetic resonance imaging (MRI) can examine muscle quality and size,detect any fatty replacement of muscle tissue, and determine whether anerve fiber has sustained compression damage. The MRI equipment createsa strong magnetic field around the body. Radio waves are then passedthrough the body to trigger a resonance signal that can be detected atdifferent angles within the body. A computer processes this resonanceinto either a three-dimensional picture or a two-dimensional “slice” ofthe scanned area.

Electromyography (EMG) involves inserting a fine needle into a muscle tocompare the amount of electrical activity present when muscles are atrest and when they contract. EMG tests can help differentiate betweenmuscle and nerve disorders.

Nerve conduction velocity (NCV) tests can precisely measure the degreeof damage in larger nerve fibers, revealing whether symptoms are beingcaused by degeneration of the myelin sheath or the axon. During thistest, a probe electrically stimulates a nerve fiber, which responds bygenerating its own electrical impulse. An electrode placed further alongthe nerve's pathway measures the speed of impulse transmission along theaxon. Slow transmission rates and impulse blockage tend to indicatedamage to the myelin sheath, while a reduction in the strength ofimpulses is a sign of axonal degeneration.

Nerve biopsy involves removing and examining a sample of nerve tissue,most often from the lower leg. Although this test can provide valuableinformation about the degree of nerve damage, it is an invasiveprocedure that is difficult to perform and may itself cause neuropathicside effects.

Skin biopsy is a test in which doctors remove a thin skin sample andexamine nerve fiber endings. Unlike NCV, it can reveal damage present insmaller fibers; in contrast to conventional nerve biopsy, skin biopsy isless invasive, has fewer side effects, and is easier to perform.

Methods of monitoring an individual for demyelination or remyelinationare known in the art. Monitoring a subject (e.g., a human patient) forremyelination, as defined herein, means evaluating the subject for achange, e.g., an improvement in one or more parameters that areindicative of remyelination, e.g., one can monitor improvement in one ormore symptoms of a demyelinating disorder. Such symptoms include any ofthe symptoms of a demyelinating disorder described herein. Remyelinationcan also be monitored by methods which include direct determination ofthe state of myelin in the subject, e.g., one can measure white mattermass using magnetic resonance imaging (MRI) or measure the thickness ofmyelin fibers using a magnetic resonance spectroscopy (MRS) brain scan.

In some embodiments, the evaluation is performed at least 1 hour, e.g.,at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2 days, 3days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11, days,12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days,or 20 days or more, or at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 12 weeks, 13 weeks,14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks ormore, or any combination thereof, after an administration, preferablythe first administration, of the polyclonal IgG. The subject can beevaluated in one or more of the following periods: prior to beginning oftreatment; during the treatment; or after one or more elements of thetreatment have been administered. Evaluating can include evaluating theneed for further treatment, e.g., evaluating whether a dosage, frequencyof administration, or duration of treatment should be altered. It canalso include evaluating the need to add or drop a selected therapeuticmodality, e.g., adding or dropping any of the treatments fordemyelinating disorders described herein. For example, continuedadministration of the polyclonal IgG could be done with one or moreadditional therapeutic agents where necessary. In a preferredembodiment, if a preselected outcome of the evaluation is obtained, anadditional step is taken, e.g., the subject is administered anothertreatment or another evaluation or test is performed. The level ofremyelination can be used to make a determination on patient care, e.g.,a selection or modification of a course of treatment or the decision ofa third party to reimburse for the treatment.

In some embodiments, monitoring a subject (e.g., a human patient) forremyelination can also include monitoring for a reduction in the size ornumber of inflammatory lesions (i.e., scleroses) using, e.g., MagneticResonance Imaging (MRI) scans, Positron-Emission Tomography (PET) scans,Diffusion-Weighted Imaging (DW-I, or DW-MRI), Diffusion Tensor Imaging,Myelography, Magnetization Transfer. In some embodiments, monitoring asubject for remyelination can include the detection of, e.g., (i)abnormal proteins such as tiny fragments of myelin, (ii) elevated levelsof or specific types of lymphocytes, and/or (iii) abnormal levels ofimmunoglobulin (IgG) molecules. In other embodiments, monitoring asubject for remyelination can include assessment of a change in thesubject's neuropsychology (e.g., the status of various abilities such asmemory, arithmetic, attention, judgment and reasoning). In someembodiments, the monitoring of a subject (e.g., a human patient) forremyelination can involve testing a patient's urine for a decrease inlevels of myelin basic protein-like material (MBP-like material), whichsubstance becomes elevated as axonal damage occurs during diseaseprogression. In some embodiments, where the demyelinating disorderaffects a subject's eyes or vision, the monitoring of a subject forremyelination can involve testing for improvements in, e.g., colorblindness.

Provided herein are methods of evaluating a subject, to determine, e.g.,if a subject is responding or not responding to a treatment for ademyelinating disorder, e.g., a therapy that increases remyelination ina subject such as administering a polyclonal IgG. The method includesproviding a reference value (e.g., a pre-administration value) for thelevel or state of myelin in the subject, and optionally, administeringto the subject a medicament that increases remyelination (e.g., apolyclonal IgG). In embodiments where a medicament is administered, themethod also includes providing a post-administration value for the levelor state of myelin in the subject (e.g., the level or state of myelinfollowing administration of a remyelination therapy) and comparing thepost-administration value with the reference value, thereby evaluatingthe subject, e.g., determining if the subject is responding or notresponding to the therapy. The post-administration value (i.e., thevalue corresponding to the state or level of myelin in a subjectfollowing a remyelination therapy) can be determined, e.g., by any ofthe assessment methods described herein. The reference value (i.e., thestate or level of myelin in a subject prior to treatment with aremyelination therapy) can also be determined, e.g., by any of theassessment methods described herein.

In some embodiments, a determination that a subject is respondingindicates that a shorter duration of treatment can/should/will be/isadministered to the subject (e.g., shorter than the treatment which isrecommended for a subject who is not responding to a therapy, or aduration shorter than currently used with existing therapies fordemyelinating disorders, and optionally, that indication is entered intoa record.

In some embodiments, a determination that a subject is respondingindicates that a shorter duration of treatment is counter-indicated forthe subject (e.g., a duration shorter than currently used with existingtreatments for demyelinating disorders, e.g., any of the treatments fordemyelinating disorders described herein), and optionally, thatindication is entered into a record.

In some embodiments, providing a comparison of the post-administrationvalue with a reference value includes: providing a determination of apost-administration level of myelin in a subject at a first time point(e.g., wherein the first time point is 6, 7, 8, 9, 10, 11, 12, 13, 14 ormore days (e.g., 3, 4, 5, 6, 8 or more weeks (e.g., 3, 4, 6, 12 or moremonths))) after the commencement of administration of the remyelinationtherapy (e.g., polyclonal IgG); providing a determination of a referencevalue of the state or level of myelin in the subject at a second timepoint that is prior to the first time point (e.g., wherein the secondtime point is prior to, or within about 1, 2, 3, 4, or 5 days of thecommencement of, administration of a remyelination therapy (e.g.,polyclonal IgG); and providing a comparison of the post administrationlevel and reference value of a subject's myelin, wherein increasedlevels of myelin in a subject (e.g., the levels differ by no more thanabout 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about5%, about 2%, or about 1%) between the post-administration level andreference value indicates that the subject is responding.

In some embodiments, the determination of whether a patient isresponding to a therapy is made by evaluating the subject for a change,an improvement, in one or more parameters that are indicative ofremyelination, e.g., one can monitor improvement in one or more symptomsof a demyelinating disorder. Such symptoms include any of the symptomsof a demyelinating disorder described herein. Remyelination can also bemonitored by methods which include direct determination of the state ofmyelin in the subject, e.g., one can measure white matter mass usingmagnetic resonance imaging (MRI), measure the thickness of myelin fibersusing a magnetic resonance spectroscopy (MRS) brain scan, or any otherdirect measures described herein.

In another embodiment, the determination of whether a patient isresponding to a therapy can also be evaluated by any other assessment orindicia described herein, including, but not limited to, monitoring apatient for a reduction in the size or number of inflammatory lesions(i.e., scleroses) present in the patient; monitoring a patient'sendoneurial fluid for a reduction in the presence or amount of, e.g.,(i) elevated levels of or specific types of lymphocytes, and/or (ii)abnormal levels of immunoglobulin (IgG) molecules; monitoring a patientfor a positive change in neuropsychology (e.g., the status of variousabilities such as memory, arithmetic, attention, judgment andreasoning); and/or monitoring a patient's urine for a decrease in levelsof myelin basic protein-like material (MBP-like material).

In some embodiments, at least a 5% (e.g., at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 50%, at least 60%, at least 70%) improvement in one or moresymptoms of a demyelinating disorder or other above-described indiciafollowing a remyelination therapy (e.g., a therapy that inducesremyelination in a subject, e.g., a therapy such as a polyclonal IgG) issufficient to classify the patient as responding to a therapy.

IV. PREPARATION OF POLYCLONAL IGG

Immunoglobulin preparations according to the present invention can beprepared from any suitable starting materials. For example,immunoglobulin preparations can be prepared from donor serum ormonoclonal or recombinant immunoglobulins. In a typical example, bloodis collected from healthy donors. Usually, the blood is collected fromthe same species of animal as the subject to which the immunoglobulinpreparation will be administered (typically referred to as “homologous”immunoglobulins). The immunoglobulins are isolated from the blood andpurified by one or more suitable procedures, such as, for example, Cohnfractionation, ultracentrifugation, electrophoretic preparation, ionexchange chromatography, affinity chromatography, immunoaffinitychromatography, polyethylene glycol fractionation, alcoholfractionation, nanofiltration, ultrafiltration/diafiltration or thelike. (See, e.g., Cohn et al., J. Am. Chem. Soc. 68:459-75 (1946);Oncley et al., J. Am. Chem. Soc. 71:541-50 (1949); Barundern et al., VoxSang. 7:157-74 (1962); Koblet et al., Vox Sang. 13:93-102 (1967);Teschner et al. Vox Sang (92):42-55 (2007); Hoppe et al. Munch MedWochenschr (34): 1749-1752 (1967), Falksveden (Swedish Patent No.348942); Tanaka et al., Braz J Med Biol Res (33)37-30 (2000); Lebing etal., Vox Sang (84):193-201 (2003); U.S. Pat. Nos. 5,122,373 and5,177,194; PCT/US2010/036470; and PCT/US2011/038247; the disclosures ofwhich are incorporated by reference herein.)

To inactivate various viral contaminants present in plasma-derivedproducts, the clarified PptG filtrate may be subjected to a solventdetergent (S/D) treatment. Methods for the detergent treatment of plasmaderived fractions are well known in the art (for review see, Pelletier JP et al., Best Pract Res Clin Haematol. 2006; 19(1):205-42). Generally,any standard S/D treatment may be used in conjunction with the methodsprovided herein.

To further purify and concentrate IgG, cation exchange and/or anionexchange chromatography can be employed. Methods for purifying andconcentrating IgG using ion exchange chromatography are well known inthe art. For example, U.S. Pat. No. 5,886,154 describes a method inwhich a Fraction II+III precipitate is extracted at low pH (betweenabout 3.8 and 4.5), followed by precipitation of IgG using caprylicacid, and finally implementation of two anion exchange chromatographysteps. U.S. Pat. No. 6,069,236 describes a chromatographic IgGpurification scheme that does not rely on alcohol precipitation at all.PCT Publication No. WO 2005/073252 describes an IgG purification methodinvolving the extraction of a Fraction II+III precipitate, caprylic acidtreatment, PEG treatment, and a single anion exchange chromatographystep. U.S. Pat. No. 7,186,410 describes an IgG purification methodinvolving the extraction of a Fraction I+II+III or Fraction IIprecipitate followed by a single anion exchange step performed at analkaline pH. U.S. Pat. No. 7,553,938 describes a method involving theextraction of a Fraction I+II+III or Fraction II+III precipitate,caprylate treatment, and either one or two anion exchange chromatographysteps. U.S. Pat. No. 6,093,324 describes a purification methodcomprising the use of a macroporous anion exchange resin operated at apH between about 6.0 and about 6.6. U.S. Pat. No. 6,835,379 describes apurification method that relies on cation exchange chromatography in theabsence of alcohol fractionation. The disclosures of the abovepublications are hereby incorporated by reference in their entiretiesfor all purposes

To reduce the viral load of an IgG composition provided herein, thecomposition may be nanofiltered using a suitable nanofiltration device.In certain embodiments, the nanofiltration device will have a mean poresize of between about 15 nm and about 200 nm. Examples of nanofilterssuitable for this use include, without limitation, DVD, DV 50, DV 20(Pall), Viresolve NFP, Viresolve NFR (Millipore), Planova 15N, 20N, 35N,and 75N (Planova). In a specific embodiment, the nanofilter may have amean pore size of between about 15 nm and about 72 nm, or between about19 nm and about 35 nm, or of about 15 nm, 19 nm, 35 nm, or 72 nm. In apreferred embodiment, the nanofilter will have a mean pore size of about35 nm, such as an Asahi PLANOVA 35N filter or equivalent thereof. In aparticular embodiment, the IgG composition recovered from the anionexchange step is nanofiltered using a nanofilter having a pore sizebetween 30 nm and 40 nm, preferably 35±2 nm. In another preferredembodiment, the nanofilter will have a mean pore size of about 19 or 20nm, such as an Asahi PLANOVA 20N filter (19±2 nm) or equivalent thereof.In a particular embodiment, the IgG composition recovered from the anionexchange step is nanofiltered using a nanofilter having a pore sizebetween 15 nm and 25 nm, preferably 19±2 nm.

In certain embodiments, immunoglobulin is prepared from gammaglobulin-containing products produced by the alcohol fractionationand/or ion exchange and affinity chromatography methods well known tothose skilled in the art. Purified Cohn Fraction II is commonly used.The starting Cohn Fraction II paste is typically about 95 percent IgGand is comprised of the four IgG subtypes. The different subtypes arepresent in Fraction II in approximately the same ratio as they are foundin the pooled human plasma from which they are obtained. The Fraction IIis further purified before formulation into an administrable product.For example, the Fraction II paste can be dissolved in a cold purifiedaqueous alcohol solution and impurities removed via precipitation andfiltration. Following the final filtration, the immunoglobulinsuspension can be dialyzed or diafiltered (e.g., using ultrafiltrationmembranes having a nominal molecular weight limit of less than or equalto 100,000 daltons) to remove the alcohol. The solution can beconcentrated or diluted to obtain the desired protein concentration andcan be further purified by techniques well known to those skilled in theart.

Preparative steps can be used to enrich a particular isotype or subtypeof immunoglobulin. For example, protein A, protein G or protein Hsepharose chromatography can be used to enrich a mixture ofimmunoglobulins for IgG, or for specific IgG subtypes. (See generallyHarlow and Lane, Using Antibodies, Cold Spring Harbor Laboratory Press(1999); Harlow and Lane, Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory Press (1988); U.S. Pat. No. 5,180,810.)

Commercial sources of polyclonal immunoglobulins can also be used. Suchsources include but are not limited to: Kiovig® 10% IVIG (BaxterHealthcare); Gammagard Liquid® 10% IVIG (Baxter Healthcare); GammagardS/D® (Baxter Healthcare); Gammagard S/D® with less than 1 mg/mL of IgAin a 5% solution (Baxter Healthcare); Gamunex®-C, 10% (Grifols USA);Flebogamma®, 5% and 10% DIF (Grifols USA); Privigen® 10% Solution (CSLBehring); Carimune® NF or Sandoglobulin® (CSL Behring); and Hizentra®20% Liquid (CSL Behring); Octagam®, 5% and 10% IVIG (Octapharma AG);Gammanorm® 16.5% SCIG (Octapharma AG). The commercial source ofimmunoglobulin preparation for use in the methods of the presentinvention is not critical.

An alternative approach is to use fragments of antibodies withantigen-binding capability, e.g., Fab′, F(ab′)₂, Fab, Fv and rIgG. See,e.g., Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co.,Rockford, Ill.); Kuby, J., Immunology, 3.sup.rd Ed., W.H. Freeman & Co.,New York (1998). The polyclonal IgG composition of the invention mayinclude fragments of one immunoglobulin isotype, i.e. IgG, or cancontain a mixture of immunoglobulin fragments of different isotypes(e.g., IgA, IgD, IgE, IgG and/or IgM). The Fc preparation also cancontain predominantly (at least 60%, at least 75%, at least 90%, atleast 95%, or at least 99%) fragments from the IgG immunoglobulinisotype, and can contain minor amounts of the other subtypes. Forexample, an Fc preparation can contain at least at least about 75%, atleast about 90%, at least about 95%, or at least about 99% IgGfragments. In addition, the polyclonal IgG preparation can comprise asingle IgG subtype or a mixture of two or more of IgG subtypes. SuitableIgG subtypes include IgG1, IgG2, IgG3, and IgG4. In a specificembodiment, the polyclonal IgG preparation comprises IgG1 fragments.

Immunoglobulins can be cleaved at any suitable time during preparationto yield Fab, F(ab′) and/or F(ab′)₂ fragments, as applicable. A suitableenzyme for cleavage is, for example, papain, pepsin or plasmin. (See,e.g., Harlow and Lane, Using Antibodies, Cold Spring Harbor LaboratoryPress (1999); Plan and Makula, Vox Sanguinis 28:157-75 (1975).) Aftercleavage, the Fc portions can be separated from the Fab, F(ab′) and/orF(ab′)₂ fragments by, for example, affinity chromatography, ion exchangechromatography, gel filtration, or the like. In a specific example,immunoglobulins are digested with papain to separate the Fc fragmentfrom the Fab fragments. The digestion mixture is then subjected tocationic exchange chromatography to separate the Fc fragments from theFab fragments.

Immunoglobulin fragments can also be prepared from hybridomas or otherculture system which express monoclonal antibody. (See, e.g., Kohler andMilstein, Nature 256:495-97 (1975); Hagiwara and Yuasa, Hum. AntibodiesHybridomas 4:15-19 (1993); Kozbor et al., Immunology Today 4:72 (1983);Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96 (1985).) Human monoclonal antibodies can be obtained,for example, from human hybridomas (see, e.g., Cote et al., Proc. Natl.Acad. Sci. USA 80:2026-30 (1983)) or by transforming human B cells withEBV virus in vitro (see, e.g., Cole et al., supra). Monoclonalantibodies produced from hybridomas can be purified and the Fc fragmentsseparated from the Fab, F(ab′) and/or F(ab′)₂ fragments as describedherein or as known to the skilled artisan.

IgG fragments also can be produced recombinantly, such as fromeukaryotic cell culture systems. For example, a single chain Fvfragments (scFv) can be recombinantly produced by Chinese hamster ovary(CHO) cells transfected with a vector containing a DNA sequence encodingthe Fv fragments. Methods for creating such recombinant mammalian cellsare described in, for example, Sambrook and Russell, Molecular Cloning,A Laboratory Manual, 3rd ed. (Cold Spring Harbor Laboratory Press (NewYork) 2001) and Ausubel et al., Short Protocols in Molecular Biology,4th ed. (John Wiley & Sons, Inc. (New York) 1999) and are known to theskilled artisan. Recombinant immunoglobulin fragments can also beproduced in other mammalian cell lines, such as baby hamster kidney(BHK) cells. Methods of culturing recombinant cells to producerecombinant proteins are also known to the art.

A variety of other expression systems can be utilized to expressrecombinant immunoglobulins IgG fragments. These include, but are notlimited to, insect cell systems and microorganisms such as yeast orbacteria which have been transfected or transformed with an expressioncassette encoding the desired IgG fragment. In certain embodiments, themicroorganism optionally can be engineered to reproduce glycosylationpatterns of mammalian or human IgG fragments.

In certain embodiments, further preparative steps can be used in orderto render an immunoglobulin preparation safe for use in the methodsaccording to the present invention. Such steps can include, for example,treatment with solvent/detergent, pasteurization and sterilization.Additional preparative steps may be used in order to ensure the safetyof a polyclonal IgG preparation. Such preparative steps can include, forexample, enzymatic hydrolysis, chemical modification via reduction andalkylation, sulfonation, treatment with B-propiolactone, treatment atlow pH, or the like. Descriptions of suitable methods can also be foundin, for example, U.S. Pat. Nos. 4,608,254; 4,687,664; 4,640,834;4,814,277; 5,864,016; 5,639,730 and 5,770,199; Romer et al., Vox Sang.42:62-73 (1982); Romer et al., Vox Sang. 42:74-80 (1990); and Rutter, J.Neurosurg. Psychiat. 57 (Suppl.):2-5 (1994) (the disclosures of whichare incorporated by reference herein).

V. PHARMACEUTICAL COMPOSITIONS AND DOSAGES

An individual in whom administration of the polyclonal IgG as set forthherein is an effective therapeutic regimen for demyelinating peripheralneuropathy, is preferably a human, but can be any mammal Thus, as can bereadily appreciated by one of ordinary skill in the art, the methods andpharmaceutical compositions of the present invention are particularlysuited to administration to any a mammal, and including, but by no meanslimited to, domestic animals, such as feline or canine subjects, farmanimals, such as but not limited to bovine, equine, caprine, ovine, andporcine subjects, wild animals (whether in the wild or in a zoologicalgarden), research animals, such as mice, rats, rabbits, goats, sheep,pigs, dogs, cats, etc., i.e., for veterinary medical use.

It is contemplated that a pharmaceutical composition comprisingpolyclonal IgG of the present invention can be administered by a varietyof methods known in the art. The route and/or mode of administrationvary depending upon the desired results, but will typically beintravenous, intramuscular, intranasal, intraperitoneal, intra-arterial,or subcutaneous. The pharmaceutical composition can include anacceptable carrier suitable for intravenous, intramuscular,subcutaneous, parenteral, spinal or epidermal administration (e.g., byinjection or infusion).

The polyclonal IgG of this invention are useful for local or systemicadministration for prophylactic and/or therapeutic treatment. Exemplarymodes of administration include, without limitation, transdermal,subcutaneous, intra-arterial, intravenous, intranasal, intramuscular,rectal, buccal, and oral administration. The pharmaceutical compositionscan be administered in a variety of unit dosage forms depending upon themethod of administration. For example, unit dosage forms include powder,tablets, pills, capsules, suppositories, ampoules, and lozenges. It isonly necessary that the active ingredient constitute an effectiveamount, i.e., such that a suitable effective dosage will be consistentwith the dosage form employed in single or multiple unit doses. Theexact individual dosages, as well as daily dosages, will, of course, bedetermined according to standard medical principles under the directionof a physician or veterinarian. The pharmaceutical polyclonal IgGimmunoglobin compositions of this invention, when administered orally,are preferably protected from digestion. This is typically accomplishedeither by complexing the antibodies with a composition to render themresistant to acidic and enzymatic hydrolysis or by packaging theantibodies in an appropriately resistant carrier such as a vesicle, inparticular a liposome (see Langer, Science 249:1527-1533 (1990); Treatet al., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);Lopez-Berestein, ibid., pp. 317-327; see generally ibid). Means ofprotecting proteins from digestion are well known in the art.

The pharmaceutical compositions of this invention are particularlyuseful for parenteral administration, such as intravenous administrationor administration into a body cavity or lumen of an organ. Thecompositions for administration will commonly comprise a composition ofpolyclonal IgG with a pharmaceutically acceptable carrier, preferably anaqueous carrier. A variety of aqueous carriers can be used, e.g.,buffered saline and the like.

Diluents that can be used in pharmaceutical compositions (e.g.,granulates) containing the active compound adapted to be formed intotablets, dragees, capsules and pills include the following: (a) fillersand extenders, e.g., starch, sugars, mannitol and silicic acid; (b)binding agents, e.g., carboxymethyl cellulose and other cellulosederivatives, alginates, gelatine and polyvinyl pyrrolidone; (c)moisturizing agents, e.g., glycerol; (d) disintegrating agents, e.g.,agar-agar, calcium carbonate and sodium bicarbonate; (e) agents forretarding dissolution, e.g., paraffin; (f) resorption accelerators,e.g., quaternary ammonium compounds; (g) surface active agents, e.g.,cetyl alcohol, glycerol monostearate; (g) adsorptive carriers, e.g.,kaolin and bentonite; (i) lubricants, e.g., talc, calcium and magnesiumstearate and solid polyethylene glycols. The diluents to be used inpharmaceutical compositions adapted to be formed into suppositories can,for example, be the usual water-soluble diluents, such as polyethyleneglycols and fats (e.g., cocoa oil and high esters, [e.g., C₁₄-alcoholwith C₁₆-fatty acid]) or mixtures of these diluents.

The pharmaceutical compositions of the invention are sterile andgenerally free of undesirable matter. For parental administration,solutions and suspensions should be sterile, e.g., water or arachis oilcontained in ampoules and, if appropriate, blood-isotonic. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, toxicity adjusting agents andthe like, for example, sodium acetate, sodium chloride, potassiumchloride, calcium chloride, sodium lactate and the like. Theconcentration of the polyclonal IgG in these formulations can varywidely, and will be selected primarily based on fluid volumes,viscosities, patient body weight and the like in accordance with theparticular mode of administration selected and the patient's needs.

Proper fluidity of the composition can be maintained, for example, byuse of coating such as lecithin, by maintenance of required particlesize in the case of dispersion and by use of surfactants. In some cases,it is preferable to include isotonic agents, for example, sugars such assucrose, polyalcohols such as mannitol or sorbitol, and sodium chloridein the composition. Stabilizers such as nicotinamide, L-proline,L-glycine, or L-isoleucine may also be employed. Long-term absorption ofthe injectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate or gelatin.

The pharmaceutical compositions which are suspensions can contain theusual diluents, such as liquid diluents, e.g., water, ethyl alcohol,propylene glycol, surface active agents (e.g., ethoxylated isostearylalcohols, polyoxyethylene sorbitols and sorbitan esters),microcrystalline cellulose, aluminum methahydroxide, bentonite,agar-agar and tragacanth, or mixtures thereof.

The pharmaceutical compositions can also contain coloring agents andpreservatives, as well as perfumes and flavoring additions (e.g.,peppermint oil and eucalyptus oil), and sweetening agents, (e.g.,saccharin and aspartame).

The pharmaceutical compositions will generally contain from 0.5 to 90%of the active ingredient by weight of the total composition.

In addition to the monoclonal antibodies, the pharmaceuticalcompositions and medicaments can also contain other pharmaceuticallyactive compounds, e.g. steroids, anti-inflammatory agents or the like.

Any diluent in the medicaments of the present invention may be any ofthose mentioned above in relation to the pharmaceutical compositions.Such medicaments may include solvents of molecular weight less than 200as the sole diluent.

Pharmaceutical compositions of the invention can be prepared inaccordance with methods well known and routinely practiced in the art.See, e.g., Remington: The Science and Practice of Pharmacy, MackPublishing Co., 20^(th) ed., 2000; and Sustained and Controlled ReleaseDrug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., NewYork, 1978. Pharmaceutical compositions are preferably manufacturedunder GMP conditions. Typically, a therapeutically effective dose orefficacious dose of the immunoglobulin preparation is employed in thepharmaceutical compositions of the invention. The pharmaceuticalcomposition can be formulated into dosage forms by conventional methodsknown to those of skill in the art. Dosage regimens are adjusted toprovide the optimum desired response (e.g., a therapeutic response). Forexample, a single bolus may be administered, several divided doses maybe administered over time or the dose may be proportionally reduced orincreased as indicated by the exigencies of the therapeutic situation.It can be advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the subjects to be treated; each unit contains apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier.

Actual dosage levels can be varied so as to obtain an amount of theactive ingredient which is effective to achieve the desired therapeuticresponse for a particular patient without being toxic to the patient. Aphysician can start doses of the pharmaceutical composition at levelslower than that required to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved. Ingeneral, effective doses vary depending upon many different factors,including the specific disease or condition to be treated, its severity,physiological state of the patient, other medications administered, andwhether treatment is prophylactic or therapeutic.

The polyclonal IgG composition can be administered on multipleoccasions. Intervals between single dosages can be daily, weekly,biweekly, every 3 weeks, every 4 weeks, monthly or yearly. Intervals canalso be irregular as indicated by measuring therapeutic progress in thepatient. Dosage and frequency can vary depending on the half-life of theantibodies in the patient.

Alternatively, the polyclonal IgG can be delivered in a controlledrelease system. For example, the polyclonal immunoglobulins may beadministered using intravenous infusion, an implantable osmotic pump, atransdermal patch, liposomes, or other modes of administration. In oneembodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit.Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment,polymeric materials can be used (see Medical Applications of ControlledRelease, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974);Controlled Drug Bioavailability, Drug Product Design and Performance,Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J.Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al.,Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989);Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment,a controlled release system can be placed in proximity of thetherapeutic target, i.e., a site of injury in the peripheral nervoussystem, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, in Medical Applications of Controlled Release, supra, vol. 2,pp. 115-138 (1984)). Other controlled release systems are discussed inthe review by Langer (Science 249:1527-1533 (1990)).

In the case of a polyclonal IgG immunoglobulin preparation, intravenousimmunoglobulin (IVIG) is commonly used. IVIG formulations are designedfor administration by injection. Because polyclonal IgG preparationshave achieved an exceptionally high immunoglobulin concentration (e.g.10% w/v in some embodiments, 15% w/v in other embodiments, 20% w/v instill other embodiments, and up to 25% w/v in still furtherembodiments), which significantly reduces the volume for atherapeutically effective dose, the composition of the present inventionis particularly advantageous for subcutaneous and/or intramuscularadministration to a patient, as well as intravenous administration.

The term “effective amount” refers to an amount of polyclonal IgGpreparation that results in an improvement or remediation of a medicalcondition being treated in the subject (e.g., for treating peripheralnerve trauma, for treating toxin-induced peripheral neuropathy, etc.).An effective amount to be administered to the subject can be determinedby a physician with consideration of individual differences in age,weight, disease severity, route of administration (e.g., intravenous v.subcutaneous) and response to the therapy.

The dosing schedule may vary, depending on the circulation half-life,and the formulation used. The compositions are administered in a mannercompatible with the dosage formulation in the therapeutically effectiveamount. Precise amounts of active ingredient required to be administereddepend on the judgment of the practitioner and are peculiar to eachindividual.

A suitable dose of polyclonal IgG may be administered to a patientweekly, biweekly, every 3 weeks, every 4 weeks, or monthly to a subject,wherein the dose ranges from about 0.050 to 5 g/kilogram of patient bodyweight, about 0.095 to 4.7 g/kilogram of patient body weight, about0.140 to 4.4 g/kilogram of patient body weight, about 0.185 to 4.1g/kilogram of patient body weight, about 0.230 to 3.8 g/kilogram ofpatient body weight, about 0.275 to 3.5 g/kilogram of patient bodyweight, about 0.320 to 3.2 g/kilogram of patient body weight, about0.365 to 2.9 g/kilogram of patient body weight, about 0.410 to 2.6g/kilogram of patient body weight, about 0.455 to 2.3 g/kilogram ofpatient body weight, about 0.500 to 2.0 g/kilogram of patient bodyweight.

In alternative embodiments, the polyclonal IgG composition of theinvention is administered weekly, biweekly, every 3 weeks, every 4weeks, or monthly to a subject at a dose of about 0.05 to 4.9 g/kilogramof patient body weight, about 0.05 to 4.8 g/kilogram of patient bodyweight, about 0.05 to 4.7 g/kilogram of patient body weight, about 0.05to 4.6 g/kilogram of patient body weight, about 0.05 to 4.5 g/kilogramof patient body weight, about 0.05 to 4.4 g/kilogram of patient bodyweight, about 0.05 to 4.3 g/kilogram of patient body weight, about 0.05to 4.2 g/kilogram of patient body weight, about 0.05 to 4.1 g/kilogramof patient body weight, about 0.05 to 4.0 g/kilogram of patient bodyweight, about 0.05 to 3.9 g/kilogram of patient body weight, about 0.05to 3.8 g/kilogram of patient body weight, about 0.05 to 3.7 g/kilogramof patient body weight, about 0.05 to 3.6 g/kilogram of patient bodyweight, about 0.05 to 3.5 g/kilogram of patient body weight, about 0.05to 3.4 g/kilogram of patient body weight, about 0.05 to 3.3 g/kilogramof patient body weight, about 0.05 to 3.2 g/kilogram of patient bodyweight, about 0.05 to 3.1 g/kilogram of patient body weight, about 0.05to 3.0 g/kilogram of patient body weight, about 0.05 to 2.9 g/kilogramof patient body weight, about 0.05 to 2.8 g/kilogram of patient bodyweight, about 0.05 to 2.7 g/kilogram of patient body weight, about 0.05to 2.6 g/kilogram of patient body weight, about 0.05 to 2.5 g/kilogramof patient body weight, about 0.05 to 2.4 g/kilogram of patient bodyweight, about 0.05 to 2.3 g/kilogram of patient body weight, about 0.05to 2.2 g/kilogram of patient body weight, about 0.05 to 2.1 g/kilogramof patient body weight, about 0.05 to 2.0 g/kilogram of patient bodyweight, about 0.05 to 1.9 g/kilogram of patient body weight, about 0.05to 1.8 g/kilogram of patient body weight, about 0.05 to 1.7 g/kilogramof patient body weight, about 0.05 to 1.6 g/kilogram of patient bodyweight, about 0.05 to 1.5 g/kilogram of patient body weight, about 0.05to 1.4 g/kilogram of patient body weight, about 0.05 to 1.3 g/kilogramof patient body weight, about 0.05 to 1.2 g/kilogram of patient bodyweight, about 0.05 to 1.1 g/kilogram of patient body weight, about 0.05to 1.0 g/kilogram of patient body weight. Clinicians familiar with thediseases treated by IgG preparations can determine the appropriate dosefor a patient according to criteria known in the art.

In other embodiments, an IVIG product can be administered to a subjectwithin the range of about 0.2 g/kilogram of patient body weight to about4 g/kilogram patient body weight each time, and the frequency ofadministration may range from twice a week, once a week, twice a month,once a month, or once every other month. One exemplary dose range ofIVIG is between about 0.1 to about 1 or about 0.2 to about 0.8 g/kgpatient body weight, typically administered at the frequency of twice amonth or once a month. For instance, IVIG is administered to somepatients at the dose of 0.2, 0.4, 0.6, or 0.8 g/kg patient body weightaccording to a twice-a-month schedule. In other cases, IVIG isadministered at the dose of 0.2, 0.4, 0.6 or 0.8 g/kg patient bodyweight according to a once-a-month schedule.

The duration of IVIG treatment for a demyelinating peripheral neuropathycan vary: it may be as short as 3 or 6 months, or may be as long as 18months, 2 years, 5 years, or 10 years. In some cases, the IVIG treatmentmay last the remainder of a patient's natural life. Effectiveness of theIVIG treatment may be assessed during the entire course ofadministration after a certain time period, e.g., every 3 months orevery 6 months for an 18-month treatment plan. In other cases,effectiveness may be assessed every 9 or 12 months for a longertreatment course. The administration schedule (dose and frequency) maybe adjusted accordingly for any subsequent administration.

For intravenous administration, the polyclonal IgG is administered at anexemplary initial infusion rate of 0.5 mL/kg/hr (0.8 mg/kg/min) for 30minutes whereas the exemplary maintenance infusion rate would be toincrease the rate every 30 minutes if tolerated up to 5 mL/kg/hr (8mg/kg/min) Infusion times may vary depending on the dose, rate ofinfusion and tolerability.

For subcutaneous administration to individuals of 40 kg patient bodyweight and greater, an exemplary initial infusion rate is 30 mL/site at20 mL/hr/site whereas an exemplary maintenance infusion rate is 30mL/site at 20-30 mL/hr/site. For subcutaneous administration toindividuals of less than 40 kg patient body weight, an exemplary initialinfusion rate is 20-30 mL/site at 15 mL/hr/site whereas an exemplarymaintenance infusion rate is 20 mL/site at 15-20 mL/hr/site. Infusiontimes may vary depending on the dose, rate of infusion and tolerability.

In accordance with the present invention, the time needed to complete acourse of the treatment can be determined by a physician and may rangefrom as short as one day to more than a month. In certain embodiments, acourse of treatment can be from 1 to 6 months.

Methods for preparing parenterally administrable compositions will beknown or apparent to those skilled in the art and are described in moredetail in such publications as Remington's Pharmaceutical Science, 15thed., Mack Publishing Company, Easton, Pa. (1980).

VI. COMBINATION THERAPY

In some embodiments, the polyclonal IgG can be administered to a subjectas a combination therapy with another treatment, e.g., another treatmentfor a demyelinating disorder (e.g., any of the demyelinating disordersdescribed herein. For example, the combination therapy can includeadministering to the subject (e.g., a human patient) one or moreadditional agents that provide a therapeutic benefit to the subject whohas, or is at risk of developing, a demyelinating disorder. In someembodiments, the polyclonal IgG and the one or more additional agentsare administered at the same time. In other embodiments, the polyclonalIgG is administered first in time and the one or more additional agentsare administered second in time. In some embodiments, the one or moreadditional agents are administered first in time and the polyclonal IgGis administered second in time. The polyclonal IgG can replace oraugment a previously or currently administered therapy. For example,upon treating with polyclonal IgG, administration of the one or moreadditional agents can cease or diminish, e.g., be administered at lowerlevels. In other embodiments, administration of the previous therapy ismaintained. In some embodiments, a previous therapy will be maintaineduntil the level of polyclonal IgG reaches a level sufficient to providea therapeutic effect. The two therapies can be administered incombination.

In some embodiments, the individual receiving a first therapy for ademyelinating disorder, e.g., Interferon Beta 1a (Avonex), InterferonBeta 1b (Rebif), glatiramer acetate (Copaxone), mitoxantrone(Novantrone), azathiprine (Imuran), cyclophosphamide (Cytoxan orNeosar), cyclosporine (Sandimmune), methotrexate, Cladribine(Leustatin), methylprednisone (Depo-Medrol or Solu-Medrol), prednisone(Deltasone), prednisolone (Delta-Cortef), dexamethasone (Medrol orDecadron), adreno-corticotrophic hormone (ACTH), or Corticotropin(Acthar), can also be administered polyclonal IgG. In some embodiments,when the human is administered polyclonal IgG, the first therapy ishalted. In other embodiments, the human is monitored for a firstpre-selected result, e.g., an improvement in one or more symptoms of ademyelinating disorder (such as increased remyelination), e.g., any ofthe symptoms of demyelinating disorders described herein. In someembodiments, when the first pre-selected result is observed, treatmentwith polyclonal IgG is decreased or halted. In some embodiments, thehuman is then monitored for a second pre-selected result after treatmentwith polyclonal IgG is halted, e.g., a worsening of a symptom of ademyelinating disorder. When the second pre-selected result is observed,administration of the polyclonal IgG to the human is reinstated orincreased, or administration of the first therapy is reinstated, or thehuman is administered both polyclonal IgG, or an increased amount ofpolyclonal IgG, and the first therapeutic regimen.

In one embodiment, a human receiving a first therapy for a demyelinatingdisorder, who is then treated with polyclonal IgG, continues to receivethe first therapy at the same or a reduced amount. In anotherembodiment, treatment with the first therapy overlaps for a time withtreatment with polyclonal IgG, but treatment with the first therapy issubsequently halted.

In some embodiments of the invention, a therapeutically effective amountof polyclonal IgG is co-administered with an anti-inflammatory to apatient in need thereof. Anti-inflammatory agents are a well-known classof pharmaceutical agents which reduce inflammation by acting on bodymechanisms (Stedman's Medical Dictionary 26 e., Williams and Wilkins,(1995); Physicians Desk Reference 51 ed, Medical Economics, (1997)).

Anti-inflammatory agents useful with the methods of the inventioninclude Non-steroidal Anti-Inflammatory Agents (NSAIDS). NSAIDStypically inhibit the body's ability to synthesize prostaglandins.Prostaglandins are a family of hormone-like chemicals, some of which aremade in response to cell injury. Specific NSAIDS approved foradministration to humans include naproxen sodium, diclofenac, sulindac,oxaprozin, diflunisal, aspirin, piroxicam, indomethocin, etodolac,ibuprofen, fenoprofen, ketoprofen, mefenamic acid, nabumetone, tolmetinsodium, and ketorolac tromethamine

Other anti-inflammatory agents useful with the methods of the inventioninclude salicylates, such as, for example, salicyclic acid, acetylsalicylic acid, choline salicylate, magnesium salicylate, sodiumsalicylate, olsalazine, and salsalate.

Other anti-inflammatory agents useful with the methods of the inventioninclude cyclooxygenase (COX) inhibitors. COX catalyzes the conversion ofarachidonate to prostaglandin H2 (PGH2); a COX inhibitor inhibits thisreaction. COX is also known as prostaglandin H synthase, or PGHsynthase. Two Cox genes, Cox-1 and Cox-2 have been isolated in severalspecies. COX-2 is tightly regulated in most tissues and usually onlyinduced in abnormal conditions, such as inflammation, rheumatic andosteo-arthritis, kidney disease and osteoporosis. COX-1 is believed tobe constitutively expressed so as to maintain platelet and kidneyfunction and inter homeostasis. Typical COX inhibitors useful in themethods of the invention include etodolac, celebrex, meloxicam,piroxicam, nimesulide, nabumetone, and rofecoxib.

Preferred anti-inflammatory agents that can be incorporated into apolymer matrix for administration in the methods of the inventioninclude: Isonixin, Amtolmetin Guacil, Proglumetacin, Piketoprofen,Difenamizole, Epirizole, Apazone, Feprazone, Morazone, Phenylbutazone,Pipebuzone, Propyphenazone, Ramifenazone, Thiazolinobutazone, Aspirin,Benoiylate, Calcium Acetylsalicylate, Etersalate, Imidazole Salicylate,Lysine Acetylsalicylate, Morpholine Salicylate, 1-Naphthyl Salicylate,Phenyl Acetysalicylate, Ampiroxicam, Droxicam, S-Adenosylmethionine,Amixetine, Benzydamine, Bucolome, Difenpiramide, Emorfazone,Guaiazulene, Nabunetone, Nimesulide, Proquazone, Superoxide Dismutase,and Tenidap.

Anti-inflammatory agents that can be appended to a polymer foradministration in the methods of the invention include: Etofenamate,Talniflumate Terofenamate, Acemetacin, Alclofenac, Bufexamac,Cinmetacin, Clopirac, Felbinac, Penclozic Acid, Fentiazac, Ibufenac,Indomethacin, Isofezolac, Isoxepac, Lonazolac, Metiazinic Acid,Mofezolac, Oxametacine, Pirazolac, Sulindac, Tiaramide, Tolmetin,Tropesin, Zomepirac, Bumadizon, Butibufen, Fenbufen, Xenbucin Clidanac,Ketorolac, Tinoridine, Benoxaprofen, Bermoprofen, Bucloxic Acid,Fenoprofen, Flunoxaprofen, Flurbiprofen, Tbuprofen, Tbuproxam,Indoprofen, Ketoprofen, Loxoprofen, Naproxen, Oxaprozin, Pirprofen,Pranoprofen, Prodznic Acid, Suprofen, Tiaprofenic Acid, Zaltoprofen,Benzpiperylon, Mofebutazone, Oxyphenbutazone, Suxibuzone,Acetaminosalol, Parsalmide, Phenyl Salicylate, Salacetamide,Salicylsulfuric Acid, Isoxican, Lomoxicam, Piroxicam, Tenoxicam,.epsilon.-Acetamidocaproic Acid, Bendazac, .alpha.-Bisabolol,Paranyline, Perisoxal, and Zileuton.

Anti-inflammatory agents that can be incorporated into a polymerbackbone for administration in the methods of the invention include:Enfenamic Acid, Aceclofenac, Glucametacin, Alminoprofen, Caiprofen,Xinoprofen, Salsalate, 3-Amino-4-hydroxybutyric Acid, Ditazol,Fepradinol, and Oxaceprol.

Anti-inflammatory agents that possess suitable ortho functionality to beincorporated into the backbone of a polymer of formula (I) as describedherein include: Flufenamic Acid, Meclofenamic Acid, Mefenamic Acid,Niflumic Acid, Tolfenamic Acid, Amfenac, Bromfenac, Diclofenac Sodium,Etodolac, Bromosaligenin, Diflunisal, Fendosal, Getitisic Acid, GlycolSalicylate, Salicilic Acid, Mesalamine, Olsalazine, SalicylamideO-Acetic Acid, Sulfasalazine,

For any anti-inflammatory agent referred to herein by a trade name it isto be understood that either the trade name product or the activeingredient possessing anti-inflammatory activity from the product can beused. Additionally, preferred agents identified herein for incorporationinto a polymer backbone can also preferably be appended to a polymer orcan be incorporated into a polymer matrix. Preferred agents that can beappended to a polymer can also preferably be incorporated into a polymermatrix.

EXAMPLES

Examples are provided below to illustrate the present invention. Theseexamples are not meant to constrain the present invention to anyparticular application or theory of operation.

Example 1 Investigation of IVIG Effect on Schwann Cells

The direct effect of human serum-derived polyclonal immunoglobulins onSchwann cell homeostasis, differentiation, and maturation asdemonstrated through various molecular and cellular variables wasinvestigated using three models: 1) a primary rat Schwann cell culturemodel; 2) a p57kip2 suppressed Schwann cell model; and 3) a co-cultureof PNS neurons and myelinating Schwann cells.

1.1. Preparation of the Rat Schwann Cell Model 1:

In this model, naive primary Schwann cells (SCs) isolated from thesciatic nerves of newborn rats were cultured. At this stage, SCs areimmature and have not yet initiated differentiation processes. Inculture, they do not progress along their differentiation program andremain proliferative but immature, most likely due to the presence ofintrinsic differentiation inhibitors (Heinen et al., 2008a).

1.2. Preparation of p57kip2 Suppressed Schwann Cell Model 2:

The present inventors have identified the p57kip2 gene as a novelintrinsic inhibitor of myelinating glial cell differentiation,maturation and myelination. It has been demonstrated that long-termshRNA dependent suppression of the p57kip2 gene uncouples primary SCdifferentiation from axonal contact. This was revealed by cell cycleexit, altered SC morphology as well as induced myelin expression (Kuryet al., 2002; Heinen et al., 2008a; Heinen et al., 2008b). In thissecond model, p57kip2 suppressed SC was used for comparison with controltransfected cells, i.e. non-differentiating cells. This culture systemprovides the unique opportunity to observe SC differentiation andmaturation in vitro in the absence of axons in a quantitative way.

1.3. Preparation of a Co-Culture of PNS Neurons and Myelinating SchwannCells—Model 3:

In this model, myelinating neuron/SC co-cultures were generated. Culturepreparations were made from embryonic Wistar rat or C57/BL6 mouse dorsalroot ganglia containing both immature sensory neurons and Schwann cellprecursors of the PNS. This co-culture simulates the in vivo situationand offers the possibility of studying the final wrapping/myelinationprocess and whether this complex interaction can be influenced byimmunoglobulin administration. Optimization of the co-culture conditionsand preparations was done according to established protocols used in theinventors' laboratory or the protocol published from Päivãlãinen et al.,(2008) with some modifications. IVIG stimulation was performed inparallel to initiation of the myelination process with dialysedIVIG/buffer preparations. IVIG/buffer dialysis was performed againstcell culture medium without supplements. All experiments were performedwith one concentration of IVIG: 20 mg/ml. The duration of stimulationwas determined by analyzing the myelination kinetics (internodeformation) after 3 and 6 days following addition of dialysedIVIG/buffer.

1.4. Cell Morphology:

Cell morphology was investigated in model 1 (rat SCs in culture) andmodel 2 (p57kip2-suppressed SCs) for up to 9 days with stimulation by 10mg/ml and 20 mg/ml of IVIG for model 1 (to observe dose dependency) andup to 7 days stimulation (9 days transfection) for model 2. Experimentswere performed with both non-dialysed and dialysed IVIG and bufferpreparations. IVIG and buffer dialysis was performed against cellculture medium without supplements. All model 2 experiments wereperformed with one concentration of dialysed IVIG (20 mg/ml). In model2, the cell growth and differentiation kinetics was also determined bymeasuring the cell protrusion length after 3 and 7 days of stimulationwith dialysed IVIG.

1.5. Cell Death/Proliferation:

Cell death/proliferation was investigated in model 1 after 2 daysstimulation with non-dialyzed and dialyzed IVIG/buffer preparations.IVIG/buffer dialysis was performed against cell culture medium withoutsupplements. All experiments were performed with one concentration ofIVIG (20 mg/ml). Two assays for measuring cell proliferation wereemployed: immunocytochemical staining against the Ki-67 antigen andimmunocytochemical staining against BrdU. Ki-67 antigen is a nuclearprotein which serves as a cellular marker for proliferation. BrdU(bromodeoxyuridine) is a nucleotide analogue of thymidine used forlabeling of proliferating cells. Immunocytochemical staining againstcaspase-3 was employed as an apoptosis marker. Caspase-3 is a proteaseactivated in apoptotic cells and therefore used as a cell death marker.Cells were fixed after two different BrdU-pulse durations of 8 h and 24h.

1.6. Gene Expression:

Gene expression was analyzed in model 1 (rat SCs in culture—section 1.1)and model 2 (p57kip2-suppressed SCs—section 1.2) exposed for up to 9days stimulation for model 1 and 7 days stimulation (9 daystransfection) for model 2 using both non-dialyzed and dialyzedIVIG/buffer preparations. Dialysed SYNAGIS preparations were used as aIgG1 control on naïve SCs (model 1). IVIG/buffer/SYNAGIS dialysis wasperformed against cell culture medium without supplements. Allexperiments were performed with one concentration of IVIG: 20 mg/ml.Transcription of myelin genes (P₀, MBP) and Fc receptors (CD64, CD32 andCD16) were measured using real-time RT-PCR.

Example 2 Schwann Cell Responds to Incubation with IVIG

2.1. Morphology:

IVIG treatment was observed to affect Schwann cell morphology. SCscultured in the presence of 10 mg/ml IVIG, and to a larger extent, inthe presence of 20 mg/ml IVIG, appeared to have larger somata andnuclei. It is currently unclear whether this is a direct impact on SCshape and cytoskeleton or adhesion properties, a result from differentcell densities, or is reflective of discrete cell surface alterationspossibly connected to the IVIG binding site(s) on the cell surface.

Significantly accelerated growth of cellular protrusions was measuredupon stimulation with IVIG using model 2 (p57kip2 suppression). Thiseffect was observed only in the early stages of the differentiationprocess, indicating an IVIG effect on the differentiation kinetics ofthe Schwann cells. To explain, the growth of cellular protrusions is amaturation parameter which was found to be dependent on suppressedp57kip2 levels. On the other hand, no effect on actin filament assemblyand structure could be observed after IVIG stimulation as revealed byTRITC conjugated phalloidin stainings.

2.2. Cell Death/Proliferation (Model 1):

After stimulation with non-dialyzed IVIG (20 mg/ml) preparations, theproliferation rate of naïve SC was significantly reduced, as revealed byassays using proliferation markers BrdU and Ki-67. See FIGS. 1-2. TheIVIG-dependent effect on the proliferation rate was diminished with IVIGdialysis, but remained statistically significant thereafter. There iscurrently no evidence of induction of apoptosis after treatment withIVIG based on the negative staining for caspase-3.

2.3. Gene Expression:

Stimulation of non-transfected SCs (model 1) with non-dialyzed anddialyzed IVIG/buffer preparations led to slight upregulation of P₀ andstrong upregulation of the MBP genes within the first 3 days oftreatment, but not after longer incubation periods. Stimulation ofp57kip2 suppressed cells (model 2) with non-dialyzed and dialyzedIVIG/buffer preparations also led to similar results regarding myelingene expression. The expression and upregulation of both myelin geneswere significantly stronger in the p57kip2-suppressed cells than in thecontrol transfected cells. Observations of the gene regulation of Fcreceptors showed that Schwann cells express CD64 and CD32 and that longterm suppression for p57kip2 leads to significant upregulation of thesegenes. There was a detectable level of CD64 Fc receptor expression inimmature SCs. In differentiating Schwann cells (upon suppression of theintrinsic inhibitor p57kip2), CD64 levels were significantly increasedwith IVIG stimulation.

Importantly, the monoclonal IgG1 controls (Synagis, Avastin andHerceptin) showed no significant effect on myelin gene expression.Stimulation of p57kip2 suppressed cells (model 2) with non-dialysed anddialysed IVIG/buffer preparations induced myelin gene expression to asimilar extent. Again MBP expression was strongly induced upon IVIGstimulation whereas P0 expression was mildly induced by the treatment.Note that myelin gene induction could be observed during a period ofseven days of stimulation and was therefore not limited to early phases.Furthermore, the expression of the p57kip2 gene was found to encode anintrinsic inhibitor of Schwann cell differentiation and wassignificantly lowered in control transfected (non-differentiating)cells.

Observations of the gene regulation of all known Fcγ receptors showedthat Schwann cells express the CD64 Fc receptor. In differentiatingSchwann cells (model 2), CD64 levels were significantly increased incomparison to control transfected (non-differentiating) cells.Regulation of the CD64 receptor expression in response to IVIGstimulation could not be observed. Of note, effects of the non-dialysedbuffer control were observed in all the gene expression experimentsperformed. This effect was, however, diminished after dialysis. Furthergene expression analyses were therefore performed with dialysed IVIGpreparations only.

2.4. Summary of Findings:

In the first 18 months of the investigation, it was discovered thatprimary SCs respond to IVIG incubation with altered cell morphologyaccompanied by an accelerated growth of cellular protrusions in earlystages of the differentiation process. Incubation with IVIG was alsofound to reduce Schwann cell proliferation without affecting cellsurvival. Furthermore, expression of two major myelin genes, P₀ and MBP,was induced in immature as well as differentiating SCs followingstimulation with IVIG. Data shows that primary rat Schwann cells wereexpress the CD64 Fc receptor and that in differentiating Schwann cells(upon suppression of the intrinsic inhibitor p57kip2), CD64 levels weresignificantly increased with exposure to IVIG. The evidence alsoprovides strong indications for an upregulation of Fc receptors (inparticular CD64) in differentiating SCs. Furthermore, a specific bindingof the human IVIGs on the Schwann cell surface was shown.

These findings support the hypothesis that SCs might exhibit immunecompetence. Reduced proliferating rate with no signs of apoptosis aswell as the induction of myelin genes, combined with accelerated growthof cellular protrusions, suggest a promotion of the differentiationprocess in the immature SC by IVIG. These are the first in vitro resultsdemonstrating that Schwann cells are not only able to respond to butalso to specifically bind immunoglobulins and that IVIG stimulation canpromote Schwann cell precursor maturation.

Example 3 GENE EXPRESSION

For further examination of the IVIG dependent effects on differentiating(p57kip2 suppressed cells, model 2) and non-differentiating (controlsuppressed cells, model 2) Schwann cell gene expression we collected 16RNA samples from 4 independent experiments for a GeneChip Array analysis(performed by Miltenyi Biotec, Germany). Sample validation was performedby determination of expression levels of MBP, P₀, p57kip2 and CD64genes.

Statistical and functional analysis was performed. Genes that wereidentified as significantly up- or down-regulated upon treatment withIVIG are provided in Tables 1 and 2. Future aims are at further geneidentification as well as validation of the obtained results.

TABLE 1 Comparison of non-differentiating Schwann cells +/− IVIG Upregulated genes Down regulated genes after treatment after treatment(gene sequence name) (gene sequence name) Tyrp1 RGD1562551 Tyrp1 Ctnna2Col24a1 Olr832 Fat3 Phgr1 Tmem72 RGD1566220 Tesc Nedd9 Il18 Slc12a3 Mt1aArhgef9 Slc40a1 Gckr Asgr1 TC636329 LOC678704 A_64_P023581 TC609365Ptprr Bcl6b Olr749 A_64_P063062 Nebl Npas2 RGD1562545 Gpx2 Hes5 Matn1Mpzl2 A_64_P022503 Ezr Fbxo32 Cryab Pls1 Fcgr2b A_64_P094596A_64_P025678 Olig1 Sox2 Plp1

TABLE 2 Comparison of differentiating Schwann cells +/− IVIG Upregulated genes Down regulated genes after treatment after treatment(gene sequence name) (gene sequence name) ENSRNOT00000064975 XM_346212Zfp334 XR_009266 Mmp25 LOC688695 A_64_P117674 Ak3l1 A_64_P151655A_64_P163956 A_44_P386999 Olig1 Sox10 Hes5

In order to confirm the observed induction of myelin gene expression (inparticular P₀ and MBP) at protein level, we performed Western-blotanalysis on p57kip2 suppressed versus control suppressed cells (model 2)after treatment with dialysed IVIG/buffer. We could demonstrate that indifferentiating Schwann cells protein levels of P₀ and to a lesserextent of MBP were increased after IVIG treatment.

Example 4 Immune-Related Proteins

It was important to confirm direct IVIG binding to the Schwann cellsurface. Applying an anti-human Fab-specific F(ab)′₂ and anti-humanFcγ-specific F(ab)′₂ antibodies, it was shown that human immunoglobulinsin the IVIG specifically bound to the Schwann cell surface. Live Schwanncells in culture were stimulated with IVIG, washed, fixed and thenseparately stained against human Fab fragments, human Fcγ fragments oragainst both epitopes in combination of a double-staining. A specificsurface binding could be localized within the perinuclear region of thecells. These binding studies were performed with naïve Schwann cells(model 1) using IVIG and IgG1 controls (Avastin and Herceptin) as wellas with differentiating Schwann cells (model 2) using IVIG. In order toaddress the question of whether CD64 receptor protein is also expressedon the Schwann cell surface, staining experiments with two anti-CD64antibodies have been initiated.

In order to determine whether CD64 receptor protein was also expressedon the Schwann cell surface staining experiments with two anti-CD64antibodies were performed. One anti-CD64 antibody appeared to bindspecifically to the rat CD64 receptor on the Schwann cells and diffusereceptor staining was distributed over the cell surface of thenon-differentiating cells. In comparison, the receptor staining ondifferentiating cells was concentrated to the cell soma above theperinuclear region. The detected CD64 signals did not coincide with theIVIG binding signals (comparison of immunological stainings).

Example 5 Internode Formation

In order to improve efficiency and reproducibility of the in vitromyelination model (model 3), a number of experimental improvement stepsusing DRG cultures derived C57/BL6 mouse embryos were performed andestablished. To this end, the protocol according to Päiväläinen et al.(2008) was modified and can now be used to study the effects of IVIGapplication on axon/Schwann cell interactions. IVIG stimulation (20mg/ml) was performed concomitant to the initiation of the myelinationprocess using dialysed IVIG/buffer preparations.

After determination of the optimal time point for the analysis at 7 daysupon initiation of myelinisation, a statistically significant number ofIVIG stimulation experiments (n=9) were performed. In order to evaluatethe ability of immunoglobulin treatment to modulate the generation ofmyelin sheaths (internode formation), the number of internodes(normalizing to the whole number of nuclei in the co-culture) of IVIGtreated were compared to the number of internodes in controlco-cultures. Although a trend towards slightly increased internodedensities could be observed, no statistically significant difference inmyelin segment formation was detected after treatment.

Example 6 In Vivo Nerve Repair Paradigm

6.1. Summary

In order to translate in vitro findings based on primary rat Schwanncell cultures to an in vivo paradigm, chronic peripheral nerve lesionswere induced in adult rats treated with IVIG or control buffer during aso called “nerve regeneration period”. Sciatic nerves were transectedand, by means of suturing religation of nerve ends, nerve regenerationwas prevented for a period of three months. After this degenerationperiod, nerves were ligated to allow regeneration to take place and IVIGor buffer was administered (i.p. injections). Nerves were allowed toregenerate for another three months until the animals were sacrificed.

The above-described surgical approach on Schwann cells was used todetermine whether IVIG stimulation can repair the activity of injuredperipheral nerves. During the three months regeneration (and IVIG/buffertreatment) period a number of functional tests were performed on liverats. Afterwards animals were sacrificed and sciatic nerves weredissected, fixed and embedded for morphological and immunohistochemicalfuture analyses aiming at the description of Schwann cell/myelin andaxonal reactions. Preliminary results were acquired from the functionalanalyses. These preliminary findings indicate that differences betweenthe two groups (IVIG vs. buffer treated animals) exist. Specifically,IVIG treated animals displayed longer and broader footprint areas(contact zones between foot and floor) as compared to buffer treatedanimals. These footprint areas also gradually increased during thetreatment period and this was accompanied with an increased landingpressure (corresponding to the force that is used by the leg to make astep or to the pressure the foot exerts to the surface). Overall thesefirst preliminary data suggest that IVIG treated animals experience anaccelerated normalization of walking behavior and an increased strengthin their leg usage.

6.2. Methods

IVIG dependent effects on Schwann cell survival were investigated.Specifically, a previously established chronic peripheral nervedenervation model (Fu and Gordon; J Neurosci 1995) was used to study theproliferation as well as remyelination and axonal regeneration indenervated nerve segments in vivo. This in vivo model features similarnerve conditions to those observed in many human nerve pathologies. Thisin vivo model also provides the advantage of focusing on regenerativeevents only as degeneration processes (i.e., immune reactions aretemporally excluded).

For this purpose sciatic nerves of 24 adult Lewis rats were transectedand nerve regeneration was prevented by means of surturing religation ofthe nerve ends. This setup results in chronically injured and denervatednerve segments. Regeneration was prevented for the period of threemonths after nerve transaction. During this period, no functional testswere performed with the animals.

After three months of degeneration all 24 rats were exposed to a delayedsciatic nerve ligation (anastomosis) in that proximal nerve segmentswere sutured to the distal nerve segments thereby allowing nerveregeneration to take place. Note that in this chronic setup, the overallregeneration capacity was significantly reduced as compared to acutenerve lesions. During this first three months period axonal and myelindegeneration process were completed.

In a first set of experiments (study 1), the generation of anti-drugantibodies (ADA) and human IgG plasma levels after IVIG application wasstudied in healthy rats (unlesioned nerves) using ELISA tests. ADAagainst IVIGs was then monitored in lesioned and treated animals assecondary readout in study 2 (see below).

In a second set of experiments (study 2), Lewis Rats with chronicperipheral nerve lesions was treated with 1 g IVIG/kg body weight(high-dose treatment) following nerve ligation (regeneration period of 3month). IVIG application was done by means of i.p. injections once everyweek in the first month and then once every second week in the last twomonths of the regeneration phase. Control rats with nerve lesionsreceived IVIG formulation buffer injections. Control buffer treated andIVIG treated animal groups comprised of 12 adult female rats each.During the period of IVIG treatment, blood samples were collected fromthe tail vein in order to monitor ADA and to determine the half-life ofhuman IgG (see study 1). Blood plasma samples were collected everysecond week prior to treatment.

6.2. Results

In order to test the degree of recovery of function of the target organsafter religation of the nerve ends, a weekly set of functionalevaluation tests were conducted. Sensory function was evaluated bytesting the withdrawal response of toe 4 and 5 after application of apain stimulus (pinch test with a forceps). Muscle strength andregeneration of muscle fibers were analyzed using the leg spread test.These two functional tests as well as monitoring of the animals' weight(health and wellbeing parameter) were done on a weekly basis. Theanimals were further subjected to weekly to monitoring of footprints andwalking tracks (i.e., the “cat walk analysis”) to evaluate functionalrecovery of the sciatic nerves.

At the end of the study, 21 animals were left: 10 animals that receivedbuffer control injections and 11 animals treated with IVIG. All ratswere sacrificed and the regenerating peripheral nerve segments, as wellas contralateral healthy control nerves were collected for furtheranalysis. For this purpose animals were divided in three groups:

Group I consists of 4 buffer treated and 4 IVIG treated animals. Sciaticnerves segments (healthy and transected) of these animals will beprocessed for electron microscopy analysis (EM). Apart from determiningaxonal density (thus measuring regeneration efficiency) this will alsoinclude a g-ratio calculation (axonal diameter divided by the diameterof the axon and its myelin sheath) in order to determine remyelinationefficiencies. This analysis is currently ongoing. Functional evaluationdata of these animals (cat walk data, pinch-test and leg spreadbehavior) were determined and preliminary results are described below.

Group II consists of 3 buffer treated and 4 IVIG treated animals.Sciatic nerves segments (healthy and transected) of these animals willbe used for immunohistochemical stainings (IHC) against axonal, myelinand glial markers in order to determine the degree of cellularredifferentiation and regeneration. Nerves are currently processed andthis study is also ongoing. Functional evaluation data of these animals(cat walk data, pinch-test and leg spread behavior) were determined andpreliminary results are available are described below.

Group III consists of 3 buffer treated and 3 IVIG treated animals. Thetransected sciatic nerves segments of these animals displayed noanatomical regeneration signs since the anastomosis did not take place.The functional evaluation data of these animals will not be included inthe overall analysis.

A preliminary evaluation of the cat walk data indicates that differencesbetween the two groups (IVIG vs. buffer treated animals) exist. IVIGtreated animals displayed longer and broader footprint areas (contactzones between foot and floor) as compared to buffer treated animals.These footprint areas also gradually increased during the treatmentperiod and this was accompanied with an increased landing pressure(corresponding to the force that is used by the leg to make a step or tothe pressure the foot exerts to the surface). Overall this data suggestthat IVIG treated animals experience an accelerated normalization ofwalking behavior and an increased strength in their leg usage.

Example 7 Supplemental Studies to Determine the Underlying Mechanisms ofIVIG Action

To better understand the underlying mechanisms of IVIG action andmechanisms by which IVIGs promote cellular maturation, detailedmolecular/cellular investigations on stimulated Schwann cells will beperformed.

As outlined above (see 5.1), a GeneChip analysis on non-differentiatingand differentiating Schwann cells exposed to IVIG treatment wasperformed and analyzed. Based upon the newly discovered upregulated anddownregulated genes (Tables 1 and 2), further validation experimentswill be conducted using quantitative real-time RT-PCR on selected genes.If necessary and applicable, additional validations using antibodies(Western-blot, immunological stainings as well as ELISA) will beperformed. This will be particularly interesting for genes related toimmune competence. Of note, this expression analysis will not only beanalyzed in order to understand what cellular processes are most IVIGsensitive, it will most likely also serve to define additional markergenes that can be used to monitor and quantify IVIG dependent reactions.

Following establishment of a suitable in vitro myelination assay (model3), a statistically significant number of IVIG stimulation experimentswill be performed. The active time windows and to which extentimmunoglobulin treatment can modulate the generation of myelin sheaths(internode formation) will be evaluated.

Using a Cy3 conjugated anti-human Fab antibody, the specific binding ofIVIGs to Schwann cell surfaces can be demonstrated. It remains to beshown whether this is due to interaction with the CD64 Fc receptor orwhether Schwann cell-specific epitopes are recognized by Fab-mediatedbinding. For this purpose, Schwann cells (model 1) will either becontacted with Fc and F(ab)₂ fractions of papain-digested IVIG or boundIVIGs on Schwann cells will be digested with papain in situ.Furthermore, the application of a FITC-conjugated anti-human Fc antibodyin combination with Cy3 conjugated anti-human Fab antibody is expectedto result in papain sensitive stainings. Two anti-CD64 antibodies willbe applied on non-differentiating and differentiating (model 2) Schwanncells in order to determine whether CD64 is also expressed as a receptorprotein on the Schwann cell surface. In case that the IVIG binding isreally mediated via this Fc receptor, it will be expected that the CD64signals coincide with the IVIG binding (immunological stainings).Further to this end, it will be examined whether an increase in CD64protein levels can be observed as a consequence of the differentiationprocess (Western-blot).

To provide functional proof for Fc-receptor involvement, pharmacologicalinhibitors such as3-(1-Methyl-1H-indol-3-yl-methylene)-2-oxo-2,3-dihydro-1H-indole-5-sulfonamideor Ly294002 interfering with spleen tyrosine kinase (Syk) andphosphatidylinositol-3-kinase (PI3K) will be applied, respectively,prior to IVIG stimulation of naïve Schwann cells (model 1). This willindicate whether these Fc-dependent signaling components are involved inMBP induction (or appropriate marker genes identified in 1.).Furthermore, digested IVIGs will be used to stimulate cultured Schwanncells (model 1) in order to reveal whether Fc or/and Fab fractions areresponsible for IVIG specific gene regulations (MBP and other markergenes identified in the gene expression analysis). Finally,shRNA-mediated suppression of CD64 expression in Schwann cells (model 1)can be used to confirm that IVIG binding is CD64 dependent as well asresponsible for the IVIG dependent induction of MBP expression (or othermarker genes identified in the gene expression analysis).

Standard Schwann cell culture (maintenance and differentiation)conditions feature high fetal calf serum concentrations (up to 10% ofvolume). It is therefore conceivable that immunoglobulins present in theserum are diminishing IVIG-dependent Schwann cell reactions. To testthis, the serum concentration will be reduced to the lower limit neededin order to assure cell survival and differentiation, the Schwann cellsstimulated with IVIGs and MBP expression levels (models 1 and 2) as wellas morphological parameters measured (model 2).

The present inventors' recent investigations revealed that Schwann celldifferentiation is critically dependent of the histone methyltransferaseenhancer of zeste homolog 2 (EZH2; Heinen et al., in revision). Uponsuppression of EZH2 activity, cultured Schwann cells showdedifferentiation reactions similar to what is observed in nervepathologies. As part of future investigations, such dedifferentiatingSchwann cells will be stimulated with IVIGs to determine expression ofSchwann cell marker and myelin genes. The latter of which were shown tobe downregulated below control levels. It will be of interest to seewhether immunoglobulin treatment is not only able to promotedifferentiation/maturation reactions (as seen with model 2; i.e. uponsuppression of the inhibitory gene p57kip2) but can also interfere withdedifferentiation processes (such as normalization of myelin geneexpression levels).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

REFERENCES

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What is claimed is:
 1. A method of treating a trauma induceddemyelinating peripheral neuropathy, the method comprising administeringa therapeutically effective amount of polyclonal IgG to a mammaldiagnosed with said neuropathy, wherein the polyclonal IgG isadministered systemically or locally to the site of the trauma.
 2. Themethod of claim 1, wherein the mammal is human.
 3. The method of claim1, wherein an anti-inflammatory agent is co-administered with thepolyclonal IgG to the mammal.
 4. The method of claim 3, wherein theanti-inflammatory agent is adrenocorticotropic hormone.
 5. The method ofclaim 1, wherein the schedule of administration of the polyclonal IgG isweekly administration.
 6. The method of claim 1, wherein the polyclonalIgG is administered to the mammal at a dose of about 0.05 to 5 g per kgof patient body weight.
 7. The method of claim 3, wherein theanti-inflammatory agent is a corticosteroid.
 8. The method of claim 3,wherein the anti-inflammatory agent is an interferon.
 9. The method ofclaim 3, wherein the anti-inflammatory agent is glatiramer acetate. 10.The method of claim 3, wherein the anti-inflammatory agent is anon-steroidal anti-inflammatory drug.
 11. The method of claim 1, whereinthe schedule of administration of the polyclonal IgG is biweeklyadministration.
 12. The method of claim 1, wherein the schedule ofadministration of the polyclonal IgG is monthly administration.
 13. Themethod of claim 1, wherein the polyclonal IgG is administered to themammal at a dose of about 0.5 to 2 g per kg of patient body weight.